Flea protease proteins, nucleic acid molecules and uses thereof

ABSTRACT

The present invention relates to flea serine protease proteins and flea cysteine protease proteins; to flea serine protease and cysteine protease nucleic acid molecules, including those that encode such proteins; to antibodies raised against such proteins; and to compounds that inhibit flea serine protease and/or cysteine protease activities. The present invention also includes methods to obtain such proteins, nucleic acid molecules, antibodies, and inhibitors. Also included in the present invention are therapeutic compositions comprising such proteins, nucleic acid molecules, antibodies, and/or inhibitors as well as the use of such therapeutic compositions to protect a host animal from flea infestation.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 08/749,699, filed Nov. 15, 1996, which claims priority under 35U.S.C. § 120 and is a continuation-in-part of U.S. patent applicationSer. No. 08/484,211 (now U.S. Pat. No. 5,972,645, issued Oct. 26, 1999);U.S. patent application Ser. No. 08/482,130 (now U.S. Pat. No.5,962,257, issued Oct. 5, 1999); U.S. patent application Ser. No.08/485,443; and U.S. patent application Ser. No. 08/485,455 (now U.S.Pat. No. 5,712,143, issued Jan. 27, 1998), each of which was filed onJun. 7, 1995, and each of which is a continuation-in-part of U.S. patentapplication Ser. No. 09/326,773, filed Oct. 18, 1994 (now U.S. Pat. No.5,766,609, issued Jun. 16, 1998), which is a continuation-in-part ofU.S. patent application Ser. No. 07/806,482, filed Dec. 13, 1991 (nowU.S. Pat. No.5,356,622, issued Oct. 18, 1994). U.S. patent applicationSer. No. 08/749,699 also claims priority under 35 U.S.C. § 120 to U.S.patent application Ser. No. 08/326,773, ibid. and to U.S. patentapplication Ser. No. 07/806,482, U.S. patent application Ser. No.08/749,699 also clams priority under 35 U.S.C. § 119/365 to PCTapplication No. PCT/US95/14442, which published as WO 96/11706 on Apr.25, 1996. Each of the applications referred to in this section isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to novel flea protease proteins and theiruse to reduce flea infestation of animals. The present invention alsorelates to the use of anti-flea protease antibodies and other compoundsthat reduce flea protease activity to reduce flea infestation ofanimals.

BACKGROUND OF THE INVENTION

Fleas, which belong to the insect order Siphonaptera, are obligateectoparasites for a wide variety of animals, including birds andmammals. Flea infestation of animals is of health and economic concernbecause fleas are known to cause and/or transmit a variety of diseases.Fleas cause and/or carry infectious agents that cause, for example, fleaallergy dermatitis, anemia, murine typhus, plague and tapeworm. Inaddition, fleas are a problem for animals maintained as pets because theinfestation becomes a source of annoyance for the pet owner who may findhis or her home generally contaminated with fleas which feed on thepets. As such, fleas are a problem not only when they are on an animalbut also when they are in the general environment of the animal.

The medical and veterinary importance of flea infestation has promptedthe development of reagents capable of controlling flea infestation.Commonly encountered methods to control flea infestation are generallyfocussed on use of insecticides in formulations such as sprays,shampoos, dusts, dips, or foams, or in pet collars. While some of theseproducts are efficacious, most, at best, offer protection of a verylimited duration. Furthermore, many of the methods are often notsuccessful in reducing flea populations on the pet for one or more ofthe following reasons: (1) failure of owner compliance (frequentadministration is required); (2) behavioral or physiological intoleranceof the pet to the pesticide product or means of administration; and (3)the emergence of flea populations resistant to the prescribed dose ofpesticide. Additional anti-flea products include nontoxic reagents suchas insect growth regulators (IGRs), including methoprene, which mimicsflea hormones and affect flea larval development.

An alternative method for controlling flea infestation is the use offlea vaccines to be administered to animals prior to or during fleainfestation. However, despite considerable interest in developinganti-flea reagents, no flea vaccine presently exists.

SUMMARY OF THE INVENTION

The present invention relates to flea serine protease proteins, to fleaaminopeptidase proteins, and to flea cysteine protease proteins; to fleaserine protease, aminopeptidase and/or cysteine protease nucleic acidmolecules, including those that encode such proteins; to antibodiesraised against such proteins; and to compounds that inhibit flea serineprotease, aminopeptidase and/or cysteine protease activities. Thepresent invention also includes methods to obtain such proteins, nucleicacid molecules, antibodies, and inhibitors. Also included in the presentinvention are therapeutic compositions comprising such proteins, nucleicacid molecules, antibodies, and/or inhibitors as well as the use of suchtherapeutic compositions to protect a host animal from flea infestation.

One embodiment of the present invention is an isolated nucleic acidmolecule that hybridizes under stringent hybridization conditions with agene including a serine protease gene comprising a nucleic acid sequenceincluding a nucleic acid molecule including SEQ ID NO:9, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:18,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:26,SEQ ID NO:28, SEQ ID NO 29, SEQ ID MO:31, SEQ ID NO:32, SEQ ID NO:34,SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42,SEQ ID NO:43 and/or SEQ ID NO:45, and a cysteine protease genecomprising a nucleic acid molecule selected from the group consisting ofSEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:88, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93 and/or SEQ ID NO:94.

The present invention also includes a nucleic acid molecule thathybridizes under stringent hybridization conditions with a nucleic acidsequence encoding a protein comprising an amino acid sequence includingSEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22,SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36,SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:67, SEQ ID NO:68,SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73,SEQ ID NO:96, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:89, SEQID NO:92 and/or SEQ ID NO:95, or with a nucleic acid sequence that is acomplement of any of the nucleic acid sequences. A preferred nucleicacid sequence of the present invention includes a nucleic acid moleculecomprising a nucleic acid sequence including SEQ ID NO:9, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:18,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:26,SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34,SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42,SEQ ID NO:43 and SEQ ID NO:45, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:91, SEQID NO:93 and/or SEQ ID NO:94, and allelic variants thereof.

The present invention also includes an isolated protein encoded by anucleic acid molecule that hybridizes under stringent hybridizationconditions with a nucleic acid molecule having a nucleic acid sequenceencoding a protein comprising an amino acid sequence including SEQ IDNO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ IDNO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:67, SEQ ID NO:68, SEQ IDNO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ IDNO:96, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:89, SEQ ID NO:92and SEQ ID NO:95.

The present invention also relates to recombinant molecules, recombinantviruses and recombinant cells that include a nucleic acid molecule ofthe present invention. Also included are methods to produce such nucleicacid molecules, recombinant molecules, recombinant viruses andrecombinant cells.

Yet another embodiment of the present invention is a therapeuticcomposition that is capable of reducing hematophagous ectoparasiteinfestation. Such a therapeutic composition includes an excipient and aprotective compound including: an isolated protein or mimetope thereofencoded by a nucleic acid molecule that hybridizes under stringenthybridization conditions with a nucleic acid molecule having a nucleicacid sequence encoding a protein comprising an amino acid sequenceincluding SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ IDNO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ IDNO:36, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:67, SEQ IDNO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ IDNO:73, SEQ ID NO:96, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ IDNO:89, SEQ ID NO:92 and SEQ ID NO:95; an isolated nucleic acid moleculethat hybridizes under stringent hybridization conditions with a genecomprising a nucleic acid sequence including SEQ ID NO:9, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:18,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:26,SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34,SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42,SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:6, SEQ ID NO:7, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:91, SEQ IDNO:93 and SEQ ID NO:94; an isolated antibody that selectively binds to aprotein encoded by a nucleic acid molecule that hybridizes understringent hybridization conditions with a nucleic acid molecule having anucleic acid sequence encoding a protein comprising an amino acidsequence including SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ IDNO:19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ IDNO:33, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ IDNO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ IDNO:72, SEQ ID NO:73, SEQ ID NO:96, SEQ ID NO:2, SEQ ID NO:5, SEQ IDNO:8, SEQ ID NO:89, SEQ ID NO:92 and SEQ ID NO:95; an inhibitor ofprotease activity identified by its ability to inhibit the activity of aprotein encoded by a nucleic acid molecule that hybridizes understringent hybridization conditions with a nucleic acid molecule having anucleic acid sequence encoding a protein comprising an amino acidsequence including SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ IDNO:19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ IDNO:33, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ IDNO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ IDNO:72, SEQ ID NO:73, SEQ ID NO:96, SEQ ID NO:2, SEQ ID NO:5, SEQ IDNO:8, SEQ ID NO:89, SEQ ID NO:92 and SEQ ID NO:95; and a mixturethereof. Also included in the present invention is a method to reduceflea infestation, comprising the step of administering to the animal atherapeutic composition of the present invention.

Another embodiment of the present invention is a method to identify acompound capable of inhibiting flea protease activity, the methodcomprising: (a) contacting an isolated flea protease protein comprisingan amino acid sequence including SEQ ID NO:10, SEQ ID NO:13, SEQ IDNO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ IDNO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:41, SEQ IDNO:44, SEQ ID NO:67, SEQ ID NO:6 8, SEQ ID NO:69, SEQ ID NO:70, SEQ IDNO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:96, SEQ ID NO:2, SEQ IDNO:5, SEQ ID NO:8, SEQ ID NO:89, SEQ ID NO:92 and SEQ ID NO:95 with aputative inhibitory compound under conditions in which, in the absenceof said compound, the protein has proteolytic activity; and (b)determining if the putative inhibitory compound inhibits the activity.The present invention also includes a kit to to identify a compoundcapable of inhibiting flea protease activity.

The present invention also includes an isolated flea protease proteinthat cleaves an immunoglobulin, when the protein is incubated in thepresence of the immunoglobulin in about 100 microliters of about 0.2MTris-HCl for about 18 hours at about 37° C. A preferred protease proteincapable of cleaving immunoglbulin comprises an amino acid sequenceselected from the group consisting of SEQ ID NO:67, SEQ ID NO:68, SEQ IDNO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73 and SEQ IDNO:96.

Another embodiment of the present invention includes a method toidentify a compound capable of inhibiting flea immunoglobulin proteinaseprotein activity, the method comprising: (a) contacting an isolated fleaimmunoglobulin proteinase protein with a putative inhibitory compoundunder conditions in which, in the absence of the compound, the proteinhas immunoglobulin proteinase activity; and (b) determining if theputative inhibitory compound inhibits the activity.

DETAILED DESCRIPTION OF THE INVENTION

The present invention includes the use of compounds that inhibit fleaprotease activity to protect a host animal from flea infestation. Theinventors have discovered that proteases are significant components ofthe flea midgut and are good targets for immunotherapeutic and/orchemotherapeutic intervention to reduce flea burden both on the hostanimal and in the immediate (i.e., surrounding) environment of theanimal. The inventors have shown, for example, that the viability and/orfecundity of fleas consuming a blood meal is reduced when the blood mealcontains compounds that reduce flea protease activity, probably becausethe compounds interfere with flea digestion and other functions.Compounds that reduce the amount and/or activity of flea proteaseswithout substantially harming the host animal are included in thepresent invention. Such compounds include flea protease vaccines,anti-flea protease antibodies, flea protease inhibitors, and/orcompounds that suppress protease synthesis; such compounds are discussedin more detail below.

One embodiment of the present invention is a method to protect a hostanimal from flea infestation by treating the animal with a compositionthat includes a compound that reduces the protease activity of fleasfeeding (includes fleas in the process of feeding as well as fleashaving fed) from the treated animal thereby reducing the flea burden onthe animal and in the environment of the animal. It is to be noted thatthe term “a” or “an” entity refers to one or more of that entity; forexample, a compound refers to one or more compounds. As such, the terms“a” (or “an”), “one or more” and “at least one” can be usedinterchangeably herein. Thus, a composition of the present invention caninclude one or more compounds that target (reduced the activity of) oneor more proteases in the flea.

As used herein, the phrase “to protect an animal from flea infestation”refers to reducing the potential for flea population expansion on andaround the animal (i.e., reducing the flea burden). Preferably, the fleapopulation size is decreased, optimally to an extent that the animal isno longer bothered by fleas. A host animal, as used herein, is an animalfrom which fleas can feed by attaching to and feeding through the skinof the animal. Fleas, and other ectoparasites, can live on a host animalfor an extended period of time or can attach temporarily to an animal inorder to feed. At any given time, a certain percentage of a fleapopulation can be on a host animal whereas the remainder can be in theenvironment surrounding the animal (i.e., in the environment of theanimal). Such an environment can include not only adult fleas, but alsoflea eggs and/or flea larvae. The environment can be of any size suchthat fleas in the environment are able to jump onto and off of a hostanimal. As such, it is desirable not only to reduce the flea burden onan animal per se, but also to reduce the flea burden in the environmentsurrounding the animal.

In accordance with the present invention, a host animal is treated byadministering to the animal a compound of the present invention in sucha manner that the compound itself (e.g., a protease inhibitor, proteasesynthesis suppressor or anti-flea protease antibody) or a productgenerated by the animal in response to administration of the compound(e.g., antibodies produced in response to a flea protease vaccine, orconversion of an inactive inhibitor “prodrug” to an active proteaseinhibitor) ultimately enters the flea midgut. An animal is preferablytreated in such a way that the compound or product thereof enters theblood stream of the animal. Fleas are then exposed to the compound whenthey feed from the animal. For example, flea protease inhibitorsadministered to an animal are administered in such a way that theinhibitors enter the blood stream of the animal, where they can be takenup by feeding fleas. In another embodiment, when a host animal isadministered a flea protease vaccine, the treated animal mounts animmune response resulting in the production of antibodies against theprotease (anti-flea protease antibodies) which circulate in the animal'sblood stream and are taken up by fleas upon feeding. Blood taken up byfleas enters the flea midgut where compounds of the present invention,or products thereof, such as anti-flea protease antibodies, fleaprotease inhibitors, and/or protease synthesis suppressors, interactwith, and reduce proteolytic activity in the flea midgut. The presentinvention also includes the ability to reduce larval flea infestation inthat when fleas feed from a host animal that has been administered atherapeutic composition of the present invention, at least a portion ofcompounds of the present invention, or products thereof, in the bloodtaken up by the flea are excreted by the flea in feces, which issubsequently ingested by flea larvae. It is of note that flea larvaeobtain most, if not all, of their nutrition from flea feces.

In accordance with the present invention, reducing proteolytic activityin flea midguts can lead to a number of outcomes that reduce flea burdenon treated animals and their surrounding environments. Such outcomesinclude, but are not limited to, (a) reducing the viability of fleasthat feed from the treated animal, (b) reducing the fecundity of femalefleas that feed from the treated animal, (c) reducing the reproductivecapacity of male fleas that feed from the treated animal, (d) reducingthe viability of eggs laid by female fleas that feed from the treatedanimal, (e) altering the blood feeding behavior of fleas that feed fromthe treated animal (e.g., fleas take up less volume per feeding or feedless frequently), (f) reducing the viability of flea larvae, for exampledue to the feeding of larvae from feces of fleas that feed from thetreated animal and/or (g) altering the development of flea larvae (e.g.,by decreasing-feeding behavior, inhibiting growth, inhibiting (e.g.,slowing or blocking) molting, and/or otherwise inhibiting maturation toadults).

One embodiment of the present invention is a composition that includesone or more compounds that reduce the activity of one or more fleaproteases directly (e.g., an anti-flea protease antibody or a fleaprotease inhibitor) and/or indirectly (e.g., a flea protease vaccine).Suitable flea proteases to target include flea aminopeptidases, fleacarboxypeptidases and/or flea endopeptidases. Such proteases can includecytosolic and/or membrane-bound forms of a protease. Preferred fleaproteases to target include, but are not limited to, serine proteases,metalloproteases, aspartic acid proteases and/or cysteine proteases. Itis to be noted that these preferred groups of proteases includeaminopeptidases, carboxypeptidases and/or endopeptidases. Preferred fleaproteases to target include, but are not limited to, proteases thatdegrade hemoglobin, proteases involved in blood coagulation and/or lytic(anti-coagulation) pathways, proteases involved in the maturation ofpeptide hormones, proteases that inhibit complement or other host immuneresponse elements (e.g., antibodies) and/or proteases involved invitellogenesis. A number of proteases are known to those skilled in theart, including, but not limited to, aminopeptidases, such as leucineaminopeptidase and aminopeptidases B and M; astacin-likemetalloproteases; calpains; carboxypeptidases, such as carboxypeptidasesA, P and Y; cathepsins, such as cathepsins B, D, E, G, H, and L,chymotrypsins; cruzipains; meprins; papains; pepsins; renins;thermolysins and trypsins. A particularly preferred protease to targetis a protease having a proteolytic activity that, when targeted with acomposition of the present invention, reduces flea burden withoutsubstantially harming the host animal. Such a protease can be identifiedusing, for example, methods as disclosed herein.

One aspect of the present invention is the discovery that a substantialamount of the proteolytic activity found in flea midguts is serineprotease activity. Both in vitro and in vivo studies using a number ofprotease inhibitors substantiate this discovery, details of which aredisclosed in the Examples. As such a particularly preferred protease totarget is a serine protease. Examples of serine proteases, include, butare not limited to, acrosins, bromelains, cathepsin G, chymotrypsins,collagenases, elastases, factor Xa, ficins, kallikreins, papains,plasmins, Staphylococcal V8 proteases, thrombins and trypsins. In oneembodiment, a preferred flea serine protease to target includes aprotease having trypsin-like or chymotrypsin-like activity. It isappreciated by those skilled in the art that an enzyme having “like”proteolytic activity has similar activity to the referenced protease,although the exact structure of the preferred substrate cleaved maydiffer. “Like” proteases usually have similar tertiary structures astheir referenced counterparts.

Protease inhibitor studies disclosed in the Examples section alsoindicate that additional preferred proteases to target includeaminopeptidases and/or metalloproteases. Examples of such proteasesinclude exo- and endo-metalloproteases, digestive enzymes, and enzymesinvolved in peptide hormone maturation. One example of an aminopeptidasethat is also a metalloprotease is leucine aminopeptidase.

Suitable compounds to include in compositions of the present inventioninclude, but are not limited to, a vaccine comprising a flea protease (aflea protease vaccine), an antibody that selectively binds to a fleaprotease (an anti-flea protease antibody), a flea protease inhibitor (acompound other than a vaccine or an antibody that inhibits a fleaprotease), and a mixture of such compounds. As used herein, a mixturethereof refers to a combination of one or more of the cited entities.Compositions of the present invention can also include compounds tosuppress protease synthesis or maturation, such as, but not limited to,protease modulating peptides.

A preferred embodiment of the present invention is a flea proteasevaccine and its use to reduce the flea population on and around ananimal. A flea protease vaccine can include one or more proteins capableof eliciting an immune response against a flea protease and can alsoinclude other components. Preferred flea protease vaccines include aflea serine protease, a flea metalloprotease, a flea aspartic acidprotease and/or a flea cysteine protease, with flea serine protease,flea metalloprotease and/or flea aminopeptidase vaccines being morepreferred. Examples of flea protease vaccines include soluble fleamidgut preparations of the present invention as well as one or moreisolated proteins of the present invention.

One embodiment of the present invention is a soluble flea midgutpreparation. Such a preparation includes primarily components naturallypresent in the lumen of a flea midgut and, depending on the method ofpreparation, can also include one or more peripheral midgut membraneproteins. Methods to preferentially include, or exclude, membraneproteins from such a preparation are known to those skilled in the art.The present invention includes the discovery that such a preparation hasproteolytic activity, of which a substantial portion is serine proteaseactivity. Preferably at least about 70 percent of the proteolyticactivity in a soluble flea midgut soluble preparation is serine proteaseactivity, as can be indicated by the ability to inhibit at least about70 percent of the proteolytic activity with4-2-aminoethylbenzenesulfonylfluoride-hydrochloride (AEBSF). Serineprotease activity can also be identified using other known inhibitors orsubstrates. Other preferred inhibitors that can inhibit at least about70 percent of the proteolytic activity of a soluble flea midgutpreparation of the present invention include soybean trypsin inhibitor,1,3-diisopropylfluorophosphate or leupeptin.

A soluble flea midgut preparation of the present invention includesproteases that range in molecular weight from about 5 kilodaltons (kD orkDa) to about 200 kD, as determined by SDS-PAGE (sodium dodecyl sulfatepolyacrylamide gel electrophoresis), with at least a substantial portionof the serine proteases ranging in molecular weight from about 5 kD toabout 60 kD, as determined by SDS-PAGE. A substantial portion ofprotease activity in a soluble flea midgut preparation of the presentinvention has a pH activity optimum ranging from about pH 5 to about pH10, preferably an activity optimum ranging from about pH 7 to about pH9, and even more preferably an activity optimum of about pH 8. While notbeing bound by theory, such a pH optimum suggests that a largeproportion of proteases in soluble flea midgut preparations of thepresent invention are serine proteases. It is also interesting to notethat the pH of the flea midgut is also about pH 8. The findings thatproteases in soluble flea midgut preparations of the present inventionexhibit a varied pattern of inhibition by protease inhibitors of a giventype (e.g., serine protease inhibitors), as well as variances seen inmolecular weights and pH optima of the proteases, suggest that there area number of protease isoforms in such preparations.

A soluble flea midgut preparation of the present invention is preferablyprepared by a method that includes the steps of (a) disrupting a fleamidgut to produce a mixture including a liquid portion and a solidportion and (b) recovering the liquid portion to obtain a soluble fleamidgut preparation. Such a method is a simplified version of methodsdisclosed in U.S. Pat. No. 5,356,622, ibid. It is to be noted that inaccordance with the present invention, methods disclosed in U.S. Pat.No. 5,356,622, ibid. can also be used to prepare soluble flea midgutpreparations having similar proteolytic activities.

Flea midguts can be obtained (e.g., dissected from) from unfed fleas orfrom fleas that recently consumed a blood meal (i.e., blood-fed fleas).Such midguts are referred to herein as, respectively, unfed flea midgutsand fed flea midguts. Flea midguts can be obtained from either male orfemale fleas. As demonstrated in the Examples section, female fleamidguts exhibit somewhat more proteolytiic activity than do male fleamidguts. Furthermore, fed flea midguts have significantly moreproteolytic activity than do unfed flea midguts. While not being boundby theory, it is believed that blood feeding induces in flea midguts thesynthesis and/or activation of proteases as well as other factors (e.g.,enzymes, other proteins, co-factors, etc.) important in digesting theblood meal, as well as in neutralizing host molecules potentiallydamaging to the flea (e.g., complement, immunoglobulins, bloodcoagulation factors). It is also to be appreciated that unfed fleamidguts may contain significant targets not found in fed flea midgutsand vice versa. Furthermore, although the present application focusesprimarily on flea midgut proteases, it is to be noted that the presentinvention also includes other components of soluble flea midgutpreparations of the present invention that provide suitable targets toreduce flea burden on an animal and in the environment of that animal;see also U.S. Pat. No. 5,356,622, ibid.

Methods to disrupt flea midguts in order to obtain a soluble flea midgutpreparation are known to those skilled in the art and can be selectedaccording to, for example, the volume being processed and the buffersbeing used. Such methods include any technique that promotes cell lysis,such as, but are not limited to, chemical disruption techniques (e.g.,exposure of midguts to a detergent) as well as mechanical disruptiontechniques (e.g., homogenization, sonication, use of a tissue blender orglass beads, and freeze/thaw techniques).

Methods to recover a soluble flea midgut preparation are also known tothose skilled in the art and can include any method by which the liquidportion of disrupted flea midguts is separated from the solid portion(e.g., filtration or centrifugation). In a preferred embodiment,disrupted flea midguts are subjected to centrifugation, preferably at anacceleration ranging from about 10,000×g to about 15,000×g for severalminutes (e.g., from about 1 min. to about 15 min.). The supernatant fromsuch a centrifugation comprises a soluble flea midgut preparation of thepresent invention.

The present invention also includes an isolated protein that includes anamino acid sequence encoded by a nucleic acid molecule capable ofhybridizing under stringent conditions (i.e., that hybridize understringent hybridization conditions) with a nucleic acid molecule thatencodes a protease present (i.e., the nucleic acid molecules hybridizewith the nucleic acid strand that is complementary to the coding strand)in (i.e., can be found in) a flea midgut, such as a midgut from ablood-fed female flea, a midgut from a blood-fed male flea, a midgutfrom an unfed female flea or a midgut from an unfed male flea. Apreferred midgut protease is present in the lumen of the midgut.

An isolated protein of the present invention, also referred to herein asan isolated protease protein, preferably is capable of eliciting animmune response against a flea midgut protease and/or has proteolyticactivity. According to the present invention, an isolated, orbiologically pure, protein, is a protein that has been removed from itsnatural milieu. As such, “isolated” and “biologically pure” do notnecessarily reflect the extent to which the protein has been purified.An isolated protease protein can be obtained from its natural source.Such an isolated protein can also be produced using recombinant DNAtechnology or chemical synthesis.

As used herein, an isolated protein of the present invention can be afull-length protein or any homologue of such a protein, such as aprotein in which amino acids have been deleted (e.g., a truncatedversion of the protein, such as a peptide), inserted, inverted,substituted and/or derivatized (e.g., by glycosylation, phosphorylation,acetylation, myristoylation, prenylation, palmitoylation, amidationand/or addition of glycerophosphatidyl inositol) such that the homologuecomprises a protein having an amino acid sequence that is sufficientlysimilar to a natural flea midgut protease that a nucleic acid sequenceencoding the homologue is capable of hybridizing under stringentconditions to (i.e., with) the complement of a nucleic acid sequenceencoding the corresponding natural flea midgut protease amino acidsequence. As used herein, stringent hybridization conditions refer tostandard hybridization conditions under which nucleic acid molecules,including oligonucleotides, are used to identify similar nucleic acidmolecules. Such standard conditions are disclosed, for example, inSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Labs Press, 1989; Sambrook et al., ibid., is incorporated byreference herein in its entirety. Stringent hybridization conditionstypically permit isolation of nucleic acid molecules having at leastabout 70k nucleic acid sequence identity with the nucleic acid moleculebeing used to probe in the hybridization reaction. Formulae to calculatethe appropriate hybridization and wash conditions to achievehybridization permitting 30t or less mismatch of nucleotides aredisclosed, for example, in Meinkoth et al., 1984, Anal. Biochem. 138,267-284; Meinkoth et al., ibid., is incorporated by reference herein inits entirety.

The minimal size of a protein homologue of the present invention is asize sufficient to be encoded by a nucleic acid molecule capable offorming a stable hybrid with the complementary sequence of a nucleicacid molecule encoding the corresponding natural protein. As such, thesize of the nucleic acid molecule encoding such a protein homologue isdependent on nucleic acid composition and percent homology between thenucleic acid molecule and complementary sequence as well as uponhybridization conditions per se (e.g., temperature, salt concentration,and formamide concentration). The minimal size of such nucleic acidmolecules is typically at least about 12 to about 15 nucleotides inlength if the nucleic acid molecules are GC-rich and at least about 15to about 17 bases in length if they are AT-rich. As such, the minimalsize of a nucleic acid molecule used to encode a protease proteinhomologue of the present invention is from about 12 to about 18nucleotides in length. There is no limit, other than a practical limit,on the maximal size of such a nucleic acid molecule in that the nucleicacid molecule can include a portion of a gene, an entire gene, ormultiple genes, or portions thereof. Similarly, the minimal size of aprotease protein homologue of the present invention is from about 4 toabout 6 amino acids in length, with preferred sizes depending on whethera full-length, multivalent (i.e., fusion protein having more than onedomain each of which has a function), or functional portions of suchproteins are desired. Protease protein homologues of the presentinvention preferably have protease activity and/or are capable ofeliciting an immune response against a flea midgut protease.

A protease protein homologue of the present invention can be the resultof allelic variation of a natural gene encoding a flea protease. Anatural gene refers to the form of the gene found most often in nature.Protease protein homologues can be produced using techniques known inthe art including, but not limited to, direct modifications to a geneencoding a protein using, for example, classic or recombinant DNAtechniques to effect random or targeted mutagenesis. Isolated proteaseproteins of the present invention, including homologues, can beidentified in a straight-forward manner by the proteins' ability toeffect proteolytic activity and/or to elicit an immune response againsta flea midgut protease. Such techniques are known to those skilled inthe art.

A preferred protease protein of the present invention is a flea serineprotease, a flea metalloprotease, a flea aspartic acid protease, a fleacysteine protease, or a homologue of any of these proteases. A morepreferred protease protein is a flea serine protease, a fleametalloprotease or a homologue of either. Also preferred is a fleaaminopeptidase or a homologue thereof. Also preferred is a flea cysteineprotease or a homologue thereof. Particularly preferred is a flea serineprotease or a homologue thereof.

Preferred protease proteins of the present invention are flea proteaseproteins having molecular weights ranging from about 5 kD to about 200kD, as determined by SDS-PAGE, and homologues of such proteins. Morepreferred are flea protease proteins having molecular weights rangingfrom about 5 kD to about 60 kD, as determined by SDS-PAGE, andhomologues of such proteins. Even more preferred are flea serineprotease proteins, particularly those having molecular weights of about26 kD (denoted PfSP26, now denoted PafSP-26K to distinguish from fleaPfSP26 as described in Example 26), about 24 kD (denoted PfSP24, nowdenoted PafSP-24K to distinguish from flea PfSP24 as described inExample 27), about 19 kD (denoted PfSP19, now denoted PafgP-19K todistinguish from flea PfSP19 as described in Example 32), about 6 kD(denoted PfSP6, now denoted PafSP-6K to distinguish from flea PfSP6 asdescribed in Example 11), about 31 kD (denoted PfSP28), about 25 kD(denoted PlfSP-25K1) from 1st instar larvae, about 25 kD (denotedPlfSP-25K3) from 3rd instar larvae, about 28 kD (denoted PlfSP-28K3) andabout 31 kD (denoted PlfSP-31K3), and flea aminopeptidase proteins,particularly those having molecular weights of about 95 kD (denotedPfAP-95K) as determined by SDS-PAGE, and homologues of such proteins.

One preferred embodiment of the present invention is an isolated fleaprotease protein that includes an amino acid sequence encoded by anucleic acid molecule that hybridizes under stringent hybridizationconditions with a flea serine protease gene, with a flea aminopeptidasegene or with a flea cysteine protease gene. As used herein, a fleaprotease gene includes all nucleic acid sequences related to a naturalflea protease gene such as regulatory regions that control production ofa flea protease protein encoded by that gene (such as, but not limitedto, transcription, translation or post-translation control regions) aswell as the coding region itself.

The inventors have discovered an extensive family of serine proteases,encoded by a family of serine protease genes. Such a gene family may bedue to allelic variants (i.e., genes having similar, but different,sequences at a given locus in a population of fleas) and/or to, theexistence of serine protease genes at more than one locus in the fleagenome. As such, the present invention includes flea serine proteasegenes comprising not only the nucleic acid sequences disclosed herein(e.g., genes including nucleic acid sequences SEQ ID NO:9, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:18,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:26,SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34,SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42,SEQ ID NO:43, SEQ ID NO:45, and/or nucleic acid sequences encodingproteins having amino acid sequences as disclosed herein (e.g., SEQ IDNO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ IDNO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:67, SEQ ID NO:68, SEQ IDNO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73 and/or SEQID NO:96, but also allelic variants of any of those nucleic acidsequences, as well as other nucleic acid molecules and amino acidsequences disclosed in the examples section. (It should be noted thatsince nucleic acid sequencing technology is not entirely error-free, allsequences represented herein are at best apparent (i.e., deduced)nucleic acid or amino acid sequences.)

A preferred flea cysteine protease gene includes nucleic acid sequenceSEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:88, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93 and/or SEQ ID NO:94,which encode a cysteine protease protein including SEQ ID NO:2, SEQ IDNO:5, SEQ ID NO:8, SEQ ID NO:89, SEQ ID NO:92 or SEQ ID NO:95.Additional preferred cysteine protease genes include allelic variants ofSEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:88, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93 and/or SEQ ID NO:94.

A preferred flea serine protease protein of the present invention isencoded by a nucleic acid molecule that hybridizes under stringenthybridization conditions with at least one of the following nucleic acidmolecules: nfSP18, nfSP24, nfSP28, nfSP32, nfSP33 and nfSP40. As usedherein, each of these nucleic acid molecules represent the entire codingregion of a flea serine protease gene of the present invention (at leastportions of which are also referred to by flea clone numbers, asdescribed in the Examples). Nucleic acid molecules that contain partialcoding regions or other parts of the corresponding gene are denoted bynames that include the size of those nucleic acid molecules (e.g.,nfSP40₄₂₈). Nucleic acid molecules containing apparent full lengthcoding regions for which the size is known also are denoted by namesthat include the size of those nucleic acid molecules (e.g., nfSP40₈₄₁).The production, and at least partial nucleic acid sequence, of suchnucleic acid molecules is disclosed in the Examples.

Particularly preferred serine protease proteins are encoded by a nucleicacid molecule that hybridizes under stringent hybridization conditionswith at least one of the following nucleic acid molecules: nfSP18₅₃₄,nfSP18₇₇₅, nfSP18₂₂₅, nfSP24₄₁₀, nfSP24₁₀₈₉, nfSP24₇₇₄, nfSP24₇₁₁,nfSP28₉₂₃, nfSP32₉₃₃, nfSP32₉₃₃, nfSP32₉₂₄, nfSP32₈₉₉, nfSP33₄₂₆,nfSP33₇₇₈, nfSP33₁₈₉₄, nfSP33₁₂₀₀, nfSP33₇₂₆, nfSP40₈₄₁ and/ornfSP40₇₁₇. Even more preferred serine protease proteins include thefollowing amino acid sequences: SEQ ID NO:10, SEQ ID NO:13, SEQ IDNO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ IDNO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:41, SEQ IDNO:44, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ IDNO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:89, SEQ ID NO:92, SEQ IDNO:95 and/or SEQ ID NO:96. Additional particularly preferred serineprotease proteins are encoded by allelic variants of nucleic acidmolecules encoding proteins that include the cited amino acid sequences.Also preferred are flea serine protease proteins including regions thathave at least about 50%, preferably at least about 75%, and morepreferably at least about 90% identity with flea serine proteaseproteins having amino acid sequences as cited herein.

One embodiment of the present invention is a flea serine protease thatdegrades immunoglobulin circulating in a host animal (i.e., fleaimmunoglobulin proteinase or IgGase). An example of a fleaimmunoglobulin proteinase is presented in the Examples section.Preferably, an immunoglobulin proteinase of the present inventioncleavesan immunoglobulin when the protein is incubated in the presence of theimmunoglobulin in about 100 microliters of about 0.2M Tris-HCl for about18 hours at about 37° C. Suitable immunoglobulin proteinase proteins ofthe present invention are capable of cleaving the hinge region of animmunoglobulin heavy chain. The hinge region of an immunoglobulin is theflexible domain that joins the Fab arms of the immunoglobulin to the Fcportion of the molecule. A more preferred immunoglobulin proteinaseprotein includes a protein having a molecular weight ranging from about25 kD to about 35 kD and more preferably having a molecular weight ofabout 31 kD, in its mature form. An even more preferred immunoglobulinproteinase protein includes a protein comprising an amino acid sequenceincluding SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ IDNO:71, SEQ ID NO:72, SEQ ID NO:73 and/or SEQ ID NO:96, which can beencoded by a gene comprising nucleic acid sequence SEQ ID NO:66. Withoutbeing bound by theory, the proteinase activity of an immunoglobulinproteinase of the present invention cleaves an immunoglobulin in such amanner that the immunoglobulin maintains intact heavy and light chainpairs, either as two Fab fragments or one F(ab′)₂ fragment. As usedherein, a Fab fragment refers to complete immunoglobulin light chainspaired with the variable region and CHl domains of an immunoglobulinheavy chain. As used herein, a F(ab′)₂ fragment refers to two Fabfragments that remain linked by a disulfide bond. Both Fab and F(ab′)₂fragments are capable of binding antigen.

A preferred flea cysteine protease protein of the present invention isencoded by a nucleic acid molecule that hybridizes under stringenthybridization conditions with nucleic acid molecule nfCP1 (a fleacysteine protease full-length coding region that includes nfCP1₅₇₃ ornfCP1₁₁₀₉ (the production of which are described in the Examples). Evenmore preferred is a cysteine protease that includes amino acid sequenceSEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:89, SEQ ID NO:92, SEQID NO:95, or a cysteine protease encoded by an allelic variant of anucleic acid molecule that includes SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:88, SEQ ID NO:90, SEQ IDNO:91, SEQ ID NO:93 or SEQ ID NO:94. Also preferred is a flea cysteineprotease protein including regions that have at least about 50%,preferably at least about 75%, and more preferably at least about 90%identity with SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:89, SEQID NO:92 or SEQ ID NO:95.

One embodiment of the present invention is an isolated protein havingproteolytic activity that is substantially inhibited by a serineprotease inhibitor, an aminopeptidase inhibitor and/or a cysteineprotease inhibitor. Such inhibition can be measured by techniques knownto those skilled in the art. To be substantially inhibited means, forexample, for a serine protease, that at least half of the proteolyticactivity of the protease protein is inhibited by a serine proteaseinhibitor. Preferably at least about 70 percent, and even morepreferably at least about 90 percent of the proteolytic activity of theprotease protein is inhibited by a serine protease inhibitor. Preferredserine protease inhibitors include flea serpin proteins, and peptides oranalogs thereof.

An isolated protein of the present invention can be produced in avariety of ways, including recovering such a protein from a flea midgutand producing such a protein recombinantly. In one embodiment, a fleamidgut protease can be recovered by methods heretofore disclosed forobtaining a soluble flea midgut preparation. A flea midgut proteaseprotein can be further purified from a disrupted flea midgut by a numberof techniques known to those skilled in the art, including, but notlimited to, affinity chromatography, ion exchange chromatography,filtration, electrophoresis (e.g., standard, capillary and flow-throughelectrophoresis), hydrophobic interaction chromatography, gel filtrationchromatography, reverse phase chromatography, concanavalin Achromatography, chromatofocusing and differential solubilization. In oneembodiment, a flea midgut protease is purified using protease inhibitoraffinity chromatography, an example of which is disclosed in theExamples section.

Another embodiment of the present invention is a method to produce anisolated protein of the present invention using recombinant DNAtechnology. Such a method includes the steps of (a) culturing arecombinant cell comprising a nucleic acid molecule encoding a proteinof the present invention to produce the protein and (b) recovering theprotein therefrom. Details on producing recombinant cells and culturingthereof are presented below. The phrase “recovering the protein” referssimply to collecting the whole fermentation medium containing theprotein and need not imply additional steps of separation orpurification. Proteins of the present invention can be purified using avariety of standard protein purification techniques, as heretoforedisclosed.

Isolated proteins of the present invention are preferably retrieved in“substantially pure” form. As used herein, “substantially pure” refersto a purity that allows for the effective use of the protein as avaccine. A vaccine for animals, for example, should exhibit nosubstantial toxicity and should be capable of stimulating the productionof antibodies in a vaccinated animal.

Another embodiment of the present invention in an isolated nucleic acidmolecule capable of hybridizing under stringent conditions with a geneencoding a flea protease present in a flea midgut. Such a nucleic acidmolecule is also referred to herein as a flea protease nucleic acidmolecule. Particularly preferred is an isolated nucleic acid moleculethat hybridizes under stringent conditions with a flea serine proteasegene, with a flea aminopeptidase gene or with a flea cysteine proteasegene. The characteristics of such genes are disclosed herein. Inaccordance with the present invention, an isolated nucleic acid moleculeis a nucleic acid molecule that has been removed from its natural milieu(i.e., that has been subject to human manipulation). As such, “isolated”does not reflect the extent to which the nucleic acid molecule has beenpurified. An isolated nucleic acid molecule can include DNA, RNA, orderivatives of either DNA or RNA.

As stated above, a flea protease gene includes all nucleic acidsequences related to a natural flea protease gene such as regulatoryregions that control production of a flea protease protein encoded bythat gene (such as, but not limited to, transcription, translation orpost-translation control regions) as well as the coding region itself. Anucleic acid molecule of the present invention can be an isolatednatural flea protease nucleic acid molecule or a homologue thereof. Anucleic acid molecule of the present invention can include one or moreregulatory regions, full-length or partial coding regions, orcombinations thereof. The minimal size of a flea protease nucleic acidmolecule of the present invention is the minimal size capable of forminga stable hybrid under stringent hybridization conditions with acorresponding natural gene. Flea protease nucleic acid molecules canalso include a nucleic acid molecule encoding a hybrid protein, a fusionprotein, a multivalent protein or a truncation fragment.

An isolated nucleic acid molecule of the present invention can beobtained from its natural source either as an entire (i.e., complete)gene or a portion thereof capable of forming a stable hybrid with thatgene. As used herein, the phrase “at least a portion of” an entityrefers to an amount of the entity that is at least sufficient to havethe functional aspects of that entity. For example, at least a portionof a nucleic acid sequence, as used herein, is an amount of a nucleicacid sequence capable of forming a stable hybrid with the correspondinggene under stringent hybridization conditions.

An isolated nucleic acid molecule of the present invention can also beproduced using recombinant DNA technology (e.g., polymerase chainreaction (PCR) amplification, cloning) or chemical synthesis. Isolatedflea protease nucleic acid molecules include natural nucleic acidmolecules and homologues thereof, including, but not limited to, naturalallelic variants and modified nucleic acid molecules in whichnucleotides have been inserted, deleted, substituted, and/or inverted insuch a manner that such modifications do not substantially interferewith the nucleic acid molecule's ability to encode a flea proteaseprotein of the present invention or to form stable hybrids understringent conditions with natural nucleic acid molecule isolates.

A flea protease nucleic acid molecule homologue can be produced using anumber of methods known to those skilled in the art (see, for example,Sambrook et al., ibid.). For example, nucleic acid molecules can bemodified using a variety of techniques including, but not limited to,classic mutagenesis techniques and recombinant DNA techniques, such assite-directed mutagenesis, chemical treatment of a nucleic acid moleculeto induce mutations, restriction enzyme cleavage of a nucleic acidfragment, ligation of nucleic acid fragments, polymerase chain reaction(PCR) amplification and/or mutagenesis of selected regions of a nucleicacid sequence, synthesis of oligonucleotide mixtures and ligation ofmixture groups to “build” a mixture of nucleic acid molecules andcombinations thereof. Nucleic acid molecule homologues can be selectedfrom a mixture of modified nucleic acids by screening for the functionof the protein encoded by the nucleic acid (e.g., the ability of ahomologue to elicit an immune response against a flea protease and/or tohave proteolytic activity) and/or by hybridization with isolated fleaprotease nucleic acids under stringent conditions.

An isolated flea protease nucleic acid molecule of the present inventioncan include a nucleic acid sequence that encodes at least one fleaprotease protein of the present invention, examples of such proteinsbeing disclosed herein. Although the phrase “nucleic acid molecule”primarily refers to the physical nucleic acid molecule and the phrase“nucleic acid sequence” primarily refers to the sequence of nucleotideson the nucleic acid molecule, the two phrases can be usedinterchangeably, especially with respect to a nucleic acid molecule, ora nucleic acid sequence, being capable of encoding an flea proteaseprotein.

One embodiment of the present invention is a flea protease nucleic acidmolecule of the present invention that is capable of hybridizing understringent conditions to a nucleic acid strand that encodes at least aportion of a flea protease or a homologue thereof or to the complementof such a nucleic acid strand. A nucleic acid sequence complement of anynucleic acid sequence of the present invention refers to the nucleicacid sequence of the nucleic acid strand that is complementary to (i.e.,can form a complete double helix with) the strand for which the sequenceis cited. It is to be noted that a double-stranded nucleic acid moleculeof the present invention for which a nucleic acid sequence has beendetermined for one strand, that is represented by a SEQ ID NO, alsocomprises a complementary strand having a sequence that is a complementof that SEQ ID NO. As such, nucleic acid molecules of the presentinvention, which can be either double-stranded or single-stranded,include those nucleic acid molecules that form stable hybrids understringent hybridization conditions with either a given SEQ ID NO denotedherein and/or with the complement of that SEQ ID NO, which may or maynot be denoted herein. Methods to deduce a complementary sequence areknown to those skilled in the art. Preferred is a flea protease nucleicacid molecule that includes a nucleic acid sequence having at leastabout 65 percent, preferably at least about 75 percent, more preferablyat least about 85 percent, and even more preferably at least about 95percent homology with the corresponding region(s) of the nucleic acidsequence encoding at least a portion of a flea protease protein.Particularly preferred is a flea protease nucleic acid molecule capableof encoding at least a portion of a flea protease that naturally ispresent in flea midguts and preferably in included in a soluble fleamidgut preparation of the present invention. Examples of nucleic acidmolecules of the present invention are disclosed in the Examplessection.

A preferred flea serine protease nucleic acid molecule of the presentinvention is a nucleic acid molecule that hybridizes under stringenthybridization conditions with at least one of the following nucleic acidmolecules: nfSP18, nfSP24, nfSP28, nfSP32, nfSP33 and/or nfSP40. Morepreferred is a nucleic acid molecule that hybridizes under stringenthybridization conditions with at least one of the following nucleic acidmolecules: nfSP18₅₃₄, nfSP18₇₇₅, nfSP18₂₂₅, nfSP24₄₁₀, nfSP24₁₀₈₉,nfSP24₇₇₄, nfSP24₇₁₁, nfSP28₇₁₁, nfSP28₉₂₃, nfSP32₉₃₃, nfSP32₉₂₄,nfSP32₆₉₉, nfSP33₄₂₆, nfSP34₇₇₈, nfSP33₁₈₉₄, nfSP33₁₂₀₀, nfSP33₇₂₆,nfSP40₈₄₁ and/or nfSP40₇₁₇, as well as other specific nucleic acidmolecules disclosed in the Examples section. Even more preferred arenucleic acid molecules that include nfSP18, nfSP24, nfSP28, nfSP32,nfSP33 and/or nfSP40 and even more nfSP18₅₃₄, nfSP18₇₇₅, nfSP18₂₂₅,nfSP24₄₁₀, nfSP24₁₀₈₉, nfSP24₇₇₄, nfSP24₇₁₁, nfSP28₉₂₃, nfSP32₉₃₃,nfSP32₉₃₃, nfSP32₉₂₄, nfSP32₆₉₉, nfSP33₄₂₆, nfSP33₇₇₈, nfSP33₁₈₉₄,nfSP33₁₂₀₀, nfSP33₇₂₆, nfSP40₈₄₁ and/or nfSP40₇₁₇, as well as otherspecific nucleic acid molecules disclosed in the Examples section.

Particularly preferred flea serine protease nucleic acid moleculesinclude at least one of the following sequences: SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ IDNO:34, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ IDNO:42, SEQ ID NO:43 and/or SEQ ID NO:45, and complements thereof, aswell as other specific nucleic acid molecules disclosed in the Examplessection. Also preferred are allelic variants of such nucleic acidmolecules.

A preferred flea cysteine protease nucleic acid molecule of the presentinvention is a nucleic acid molecule that hybridizes under stringenthybridization conditions with nfCP1₅₇₃, or nfCP1₁₁₀₉ (the production ofwhich are described in the Examples). More preferred is a cysteineprotease nucleic acid molecule that includes nfCP1₅₇₃ or nfCP1₁₁₀₉.Particularly preferred is a nucleic acid molecule that includes nucleicacid sequence SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93 and/or SEQID NO:94, or allelic variants of such nucleic acid molecules.

Knowing a nucleic acid molecule of a flea protease protein of thepresent invention allows one skilled in the art to make copies of thatnucleic acid molecule as well as to obtain a nucleic acid moleculeincluding additional portions of flea protease protein-encoding genes(e.g., nucleic acid molecules that include the translation start siteand/or transcription and/or translation control regions), and/or fleaprotease nucleic acid molecule homologues. Knowing a portion of an aminoacid sequence of a flea protease protein of the present invention allowsone skilled in the art to clone nucleic acid sequences encoding such aflea protease protein. In addition, a desired flea protease nucleic acidmolecule can be obtained in a variety of ways including screeningappropriate expression libraries with antibodies which bind to fleaprotease proteins of the present invention; traditional cloningtechniques using oligonucleotide probes of the present invention toscreen appropriate libraries or DNA; and PCR amplification ofappropriate libraries, or RNA or DNA using oligonucleotide primers ofthe present invention (genomic and/or cDNA libraries can be used). Toisolate flea protease nucleic acid molecules, preferred cDNA librariesinclude cDNA libraries made from unfed whole fleas, fed whole fleas, fedflea midguts, unfed flea midguts, and flea salivary glands. Techniquesto clone and amplify genes are disclosed, for example, in Sambrook etal., ibid. The Examples section includes examples of the isolation ofcDNA sequences encoding flea protease proteins of the present invention.

The present invention also includes nucleic acid molecules that areoligonucleotides capable of hybridizing, under stringent conditions,with complementary regions of other, preferably longer, nucleic acidmolecules of the present invention that encode at least a portion of aflea protease protein. Oligonucleotides of the present invention can beRNA, DNA, or derivatives of either. The minimal size of sucholigonucleotides is the size required to form a stable hybrid between agiven oligonucleotide and the complementary sequence on another nucleicacid molecule of the present invention. Minimal size characteristics aredisclosed herein. The size of the oligonucleotide must also besufficient for the use of the oligonucleotide in accordance with thepresent invention. Oligonucleotides of the present invention can be usedin a variety of applications including, but not limited to, as probes toidentify additional nucleic acid molecules, as primers to amplify orextend nucleic acid molecules or in therapeutic applications to inhibitflea protease production. Such therapeutic applications include the useof such oligonucleotides in, for example, antisense-, triplexformation-, ribozyme- and/or RNA drug-based technologies. The presentinvention, therefore, includes such oligonucleotides and methods tointerfere with the production of flea protease proteins by use of one ormore of such technologies.

The present invention also includes a recombinant vector, which includesa flea protease nucleic acid molecule of the present invention insertedinto any vector capable of delivering the nucleic acid molecule into ahost cell. Such a vector contains heterologous nucleic acid sequences,that is nucleic acid sequences that are not naturally found adjacent toflea protease nucleic acid molecules of the present invention. Thevector can be either RNA or DNA, either prokaryotic or eukaryotic, andtypically is a virus or a plasmid. Recombinant vectors can be used inthe cloning, sequencing, and/or otherwise manipulating of flea proteasenucleic acid molecules of the present invention. One type of recombinantvector, herein referred to as a recombinant molecule and described inmore detail below, can be used in the expression of nucleic acidmolecules of the present invention. Preferred recombinant vectors arecapable of replicating in the transformed cell. Preferred nucleic acidmolecules to include in recombinant vectors of the present invention aredisclosed herein.

As heretofore disclosed, one embodiment of the present invention is amethod to produce a flea protease protein of the present invention byculturing a cell capable of expressing the protein under conditionseffective to produce the protein, and recovering the protein. Apreferred cell to culture is a recombinant cell that is capable ofexpressing the flea protease protein, the recombinant cell beingproduced by transforming a host cell with one or more nucleic acidmolecules of the present invention. Transformation of a nucleic acidmolecule into a cell can be accomplished by any method by which anucleic acid molecule can be inserted into the cell. Transformationtechniques include, but are not limited to, transfection,electroporation, microinjection, lipofection, adsorption, and protoplastfusion. A recombinant cell may remain unicellular or may grow into atissue, organ or a multicellular organism. Transformed nucleic acidmolecules of the present invention can remain extrachromosomal or canintegrate into one or more sites within a chromosome of the transformed(i.e., recombinant) cell in such a manner that their ability to beexpressed is retained. Preferred nucleic acid molecules with which totransform a host cell are disclosed herein.

Suitable host cells to transform include any cell that can betransformed and that can express the introduced flea protease protein.Such cells are, therefore, capable of producing flea protease proteinsof the present invention after being transformed with at least onenucleic acid molecule of the present invention. Host cells can be eitheruntransformed cells or cells that are already transformed with at leastone nucleic acid molecule. Suitable host cells of the present inventioncan include bacterial, fungal (including yeast), insect, animal andplant cells. Preferred host cells include bacterial, yeast, insect andmammalian cells, with bacterial (e.g., E. coli) and insect (e.g.,Spodoptera) cells being particularly preferred.

A recombinant cell is preferably produced by transforming a host cellwith one or more recombinant molecules, each comprising one or morenucleic acid molecules of the present invention operatively linked to anexpression vector containing one or more transcription controlsequences. The phrase operatively linked refers to insertion of anucleic acid molecule into an expression vector in a manner such thatthe molecule is able to be expressed when transformed into a host cell.As used herein, an expression vector is a DNA or RNA vector that iscapable of transforming a host cell and of effecting expression of aspecified nucleic acid molecule. Preferably, the expression vector isalso capable of replicating within the host cell. Expression vectors canbe either prokaryotic or eukaryotic, and are typically viruses orplasmids. Expression vectors of the present invention include anyvectors that function (i.e., direct gene expression) in recombinantcells of the present invention, including in bacterial, fungal, insect,animal, and/or plant cells. As such, nucleic acid molecules of thepresent invention can be operatively linked to expression vectorscontaining regulatory sequences such as promoters, operators,repressors, enhancers, termination sequences, origins of replication,and other regulatory sequences that are compatible with the recombinantcell and that control the expression of nucleic acid molecules of thepresent invention. As used herein, a transcription control sequenceincludes a sequence which is capable of controlling the initiation,elongation, and termination of transcription. Particularly importanttranscription control sequences are those which control transcriptioninitiation, such as promoter, enhancer, operator and repressorsequences. Suitable transcription control sequences include anytranscription control sequence that can function in at least one of therecombinant cells of the present invention. A variety of suchtranscription control sequences are known to those skilled in the art.Preferred transcription control sequences include those which functionin bacterial, yeast, helminth, insect and mammalian cells, such as, butnot limited to, tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB,bacteriophage lambda (λ) (such as λp_(L) and λp_(R) and fusions thatinclude such promoters), bacteriophage T7, T7lac, bacteriophage T3,bacteriophage SP6, bacteriophage SP01, metallothionein, alpha matingfactor, Pichia alcohol oxidase, alphavirus subgenomic promoters (such asSindbis virus subgenomic promoters), baculovirus, Heliothis zea insectvirus, vaccinia virus, herpesvirus, poxvirus, adenovirus, simian virus40, retrovirus actin, retroviral long terminal repeat, Rous sarcomavirus, heat shock, phosphate and nitrate transcription control sequencesas well as other sequences capable of controlling gene expression inprokaryotic or eukaryotic cells. Additional suitable transcriptioncontrol sequences include tissue-specific promoters and enhancers aswell as lymphokine-inducible promoters (e.g., promoters inducible byinterferons or interleukins). Transcription control sequences of thepresent invention can also include naturally occurring transcriptioncontrol sequences naturally associated with a DNA sequence encoding aflea protease protein.

Expression vectors of the present invention may also contain secretorysignals (i.e., signal segment nucleic acid sequences) to enable anexpressed flea protease protein to be secreted from the cell thatproduces the protein. Suitable signal segments include a flea proteaseprotein signal segment or any heterologous signal segment capable ofdirecting the secretion of a flea protease protein, including fusionproteins, of the present invention. Preferred signal segments include,but are not limited to, flea protease, tissue plasminogen activator(t-PA), interferon, interleukin, growth hormone, histocompatibility andviral envelope glycoprotein signal segments.

Expression vectors of the present invention may also contain fusionsequences which lead to the expression of inserted nucleic acidmolecules of the present invention as fusion proteins. Inclusion of afusion sequence as part of a flea protease nucleic acid molecule of thepresent invention can enhance the stability during production, storageand/or use of the protein encoded by the nucleic acid molecule.Furthermore, a fusion segment can function as a tool to simplifypurification of a flea protease protein, such as to enable purificationof the resultant fusion protein using affinity chromatography. Asuitable fusion segment can be a domain of any size that has the desiredfunction (e.g., increased stability and/or purification tool). It iswithin the scope of the present invention to use one or more fusionsegments. Fusion segments can be joined to amino and/or carboxyl terminiof a flea protease protein. Linkages between fusion segments and fleaprotease proteins can be constructed to be susceptible to cleavage toenable straight-forward recovery of the flea protease proteins. Fusionproteins are preferably produced by culturing a recombinant celltransformed with a fusion nucleic acid sequence that encodes a proteinincluding the fusion segment attached to either the carboxyl and/oramino terminal end of a flea protease protein.

A recombinant molecule of the present invention is a molecule that caninclude at least one of any nucleic acid molecule heretofore describedoperatively linked to at least one of any transcription control sequencecapable of effectively regulating expression of the nucleic acidmolecule(s) in the cell to be transformed. A preferred recombinantmolecule includes one or more nucleic acid molecules of the presentinvention, with those that encode one or more flea protease proteins,and particularly one or more flea serine protease, aminopeptidase and/orcysteine protease proteins, being more preferred. Similarly, a preferredrecombinant cell includes one or more nucleic acid molecules of thepresent invention, with those that encode one or more flea proteaseproteins, and particularly one or more flea serine protease,aminopeptidase, and/or cysteine protease proteins, being more preferred.

It may be appreciated by one skilled in the art that use of recombinantDNA technologies can improve expression of transformed nucleic acidmolecules by manipulating, for example, the number of copies of thenucleic acid molecules within a host cell, the efficiency with whichthose nucleic acid molecules are transcribed, the efficiency with whichthe resultant transcripts are translated, and the efficiency ofpost-translational modifications. Recombinant techniques useful forincreasing the expression of nucleic acid molecules of the presentinvention include, but are not limited to, operatively linking nucleicacid molecules to high-copy number plasmids, integration of the nucleicacid molecules into one or more host cell chromosomes, addition ofvector stability sequences to plasmids, substitutions or modificationsof transcription control signals (e.g., promoters, operators,enhancers), substitutions or modifications of translational controlsignals (e.g., ribosome binding sites, Shine-Dalgarno sequences),modification of nucleic acid molecules of the present invention tocorrespond to the codon usage of the host cell, deletion of sequencesthat destabilize transcripts, and use of control signals that temporallyseparate recombinant cell growth from recombinant protein productionduring fermentation. The activity of an expressed recombinant protein ofthe present invention may be improved by fragmenting, modifying, orderivatizing the resultant protein.

In accordance with the present invention, recombinant cells can be usedto produce flea protease proteins of the present invention by culturingsuch cells under conditions effective to produce such a protein, andrecovering the protein. Effective conditions to produce a proteininclude, but are not limited to, appropriate media, bioreactor,temperature, pH and oxygen conditions that permit protein production. Anappropriate, or effective, medium refers to any medium in which a cellof the present invention, when cultured, is capable of producing a fleaprotease protein. Such a medium is typically an aqueous mediumcomprising assimilable carbohydrate, nitrogen and phosphate sources, aswell as appropriate salts, minerals, metals and other nutrients, such asvitamins. The medium may comprise complex nutrients or may be a definedminimal medium.

Cells of the present invention can be cultured in conventionalfermentation bioreactors, which include, but are not limited to, batch,fed-batch, cell recycle, and continuous fermentors. Culturing can alsobe conducted in shake flasks, test tubes, microtiter dishes, and petriplates. Culturing is carried out at a temperature, pH and oxygen contentappropriate for the recombinant cell. Such culturing conditions are wellwithin the expertise of one of ordinary skill in the art.

Depending on the vector and host system used for production, resultantflea protease proteins may either remain within the recombinant cell; besecreted into the fermentation medium; be secreted into a space betweentwo cellular membranes, such as the periplasmic space in E. coli; or beretained on the outer surface of a cell or viral membrane. Methods topurify such proteins are heretofore disclosed.

The present invention also includes isolated anti-flea proteaseantibodies and their use to reduce flea infestation on a host animal aswell as in the environment of the animal. An anti-flea protease antibodyis an antibody capable of selectively binding to a protease present in aflea midgut, including female and male fed midguts as well as female andmale unfed midguts. An anti-flea protease antibody preferably binds tothe protease in such a way as to reduce the proteolytic activity of thatprotease.

Isolated antibodies are antibodies that have been removed from theirnatural milieu. The term “isolated” does not refer to the state ofpurity of such antibodies. As such, isolated antibodies can includeanti-sera containing such antibodies, or antibodies that have beenpurified to varying degrees. As used herein, the term “selectively bindsto” refers to the ability of such antibodies to preferentially bind tothe protease against which the antibody was raised (i.e., to be able todistinguish that protease from unrelated components in a mixture.).Binding affinities typically range from about 10³ M⁻¹ to about 10¹² M⁻¹.Binding can be measured using a variety of methods known to thoseskilled in the art including immunoblot assays, immunoprecipitationassays, radioimmunoassays, enzyme immunoassays (e.g., ELISA),immunofluorescent antibody assays and immunoelectron microscopy; see,for example, Sambrook et al., ibid.

Antibodies of the present invention can be either polyclonal ormonoclonal antibodies. Antibodies of the present invention includefunctional equivalents such as antibody fragments andgenetically-engineered antibodies, including single chain antibodies,that are capable of selectively binding to at least one of the epitopesof the protein used to obtain the antibodies. Antibodies of the presentinvention also include chimeric antibodies that can bind to more thanone epitope. Preferred antibodies are raised in response to proteinsthat are encoded, at least in part, by a flea protease nucleic acidmolecule of the present invention.

Anti-flea antibodies of the present invention include antibodies raisedin an animal administered a flea protease vaccine of the presentinvention that exert their effect when fleas feed from the vaccinatedanimal's blood containing such antibodies. Anti-flea antibodies of thepresent invention also include antibodies raised in an animal againstone or more flea protease proteins, or soluble flea midgut preparations,of the present invention that are then recovered from the animal usingtechniques known to those skilled in the art. Yet additional antibodiesof the present invention are produced recombinantly using techniques asheretofore disclosed for flea protease proteins of the presentinvention. Antibodies produced against defined proteins can beadvantageous because such antibodies are not substantially contaminatedwith antibodies against other substances that might otherwise causeinterference in a diagnostic assay or side effects if used in atherapeutic composition.

Anti-flea protease antibodies of the present invention have a variety ofuses that are within the scope of the present invention. For example,such antibodies can be used in a composition of the present invention topassively immunize an animal in order to protect the animal from fleainfestation. Anti-flea antibodies can also be used as tools to screenexpression libraries and/or to recover desired proteins of the presentinvention from a mixture of proteins and other contaminants.Furthermore, antibodies of the present invention can be used to targetcytotoxic agents to fleas in order to kill fleas. Targeting can beaccomplished by conjugating (i.e., stably joining) such antibodies tothe cytotoxic agents using techniques known to those skilled in the art.

A preferred anti-flea protease antibody of the present invention canselectively bind to, and preferentially reduce the proteolytic activityof, a flea serine protease, a flea metalloprotease, a flea aspartic acidprotease and/or a flea cysteine protease. More preferred anti-fleaprotease antibodies include anti-flea serine protease antibodies,anti-flea metalloprotease antibodies, anti-flea aminopeptidaseantibodies, and anti-flea cysteine protease antibodies. Particularlypreferred are anti-flea serine protease antibodies, anti-fleaaminopeptidase antibodies, and anti-flea cysteine protease antibodies,including those raised against flea serine protease proteins, fleaaminopeptidase proteins or cysteine protease proteins of the presentinvention.

The present invention also includes the use of protease inhibitors thatreduce proteolytic activity of flea proteases to reduce flea infestationof animals and the surrounding environment. As used herein, proteaseinhibitors are compounds that interact directly with a protease therebyinhibiting that protease's activity, usually by binding to or otherwiseinteracting with the protease's active site. Protease inhibitors areusually relatively small compounds and as such differ from anti-proteaseantibodies that interact with the active site of a protease.

Protease inhibitors can be used directly as compounds in compositions ofthe present invention to treat animals as long as such compounds are notharmful to the animals being treated. Protease inhibitors can also beused to identify preferred types of flea proteases to target usingcompositions of the present invention. For example, the inventors haveshown herein the predominance of serine proteases in flea midguts,particularly in soluble flea midgut preparations, using proteaseinhibitors. Such knowledge suggests that effective reduction of fleainfestation of an animal can be achieved using serine protease vaccines,anti-flea serine protease antibodies and other inhibitors of serineprotease synthesis and activity that can be tolerated by the animal. Forexample, flea immunoglobulin proteinase activity disclosed herein can betargeted to reduce flea infestation. That other proteases are alsopresent in flea midguts according to the present invention also suggeststargeting such proteases. Methods to use protease inhibitors are knownto those skilled in the art; examples of such methods are disclosedherein.

In one embodiment, a protease inhibitor that can be used in acomposition of the present invention to treat an animal is identified bya method including the following steps: (a) identifying candidate (i.e.,putative, possible) inhibitor compounds by testing the efficacy of oneor more protease inhibitors (i) in vitro for their ability to inhibitflea protease activity and/or (ii) in a flea feeding assay for theirability to reduce the survival and/or fecundity of fleas by adding theinhibitors to the blood meal of a flea being maintained, for example, ina feeding system, such as that described by Wade et al., 1988, J. MedEntomol. 25, 186-190; and (b) testing the efficacy of the candidateinhibitor compounds in animals infested with fleas. Although one doesnot need both in vixtro assay data and flea feeding assay data todetermine which candidate compounds to administer to animals, evaluationof both sets of data is preferred since data from neither of the assaysnecessarily predicts data to be obtained from the other assay. Forexample, candidate compounds identified using the in vitro assay maywork “in the test tube” but may not work in vivo for a number ofreasons, including the presence of interfering components in the bloodmeal that inhibit the activity of such compounds; e.g., althoughaprotinin can inhibit at least some flea serine proteases in vitro,aprotinin does not work well in the presence of serum proteins, such asare found in the blood. Furthermore, candidate inhibitor compoundsidentified by the flea feeding assays can include not only desiredcompounds but also compounds that reduce the viability and/or fecundityof fleas due to general toxicity (e.g., affecting the mitochondria offleas).

In a preferred embodiment, an inhibitor of a flea protease of thepresent invention is identified by a method comprising: (a) contactingan isolated flea protease protein comprising an amino acid sequenceincluding SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ IDNO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ IDNO:36, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:67, SEQ IDNO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ IDNO:73, SEQ ID NO:96, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ IDNO:89, SEQ ID NO:92 and/or SEQ ID NO:95 with a putative inhibitorycompound under conditions in which, in the absence of the compound, theprotein has proteolytic activity; and (b) determining if the putativeinhibitory compound inhibits the activity. A test kit can be used toperform such method. A preferred test kit comprises an isolated fleaprotease protein comprising an amino acid sequence including SEQ IDNO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ IDNO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:67, SEQ ID NO:68, SEQ IDNO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ IDNO:96, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:89, SEQ ID NO:92and/or SEQ ID NO:95, and a means for determining the extent ofinhibition of the activity in the presence of a putative inhibitorycompound.

In another embodiment, protease inhibitors are used in the purificationof corresponding proteases by, for example, affinity chromatography, inwhich, a protease inhibitor is incubated with a mixture containing adesired protease under conditions that the inhibitor forms a complexwith the protease. The protease can then be recovered from the complex.The protease inhibitor can be attached to a solid support and/or belabelled with, for example, a radioactive, fluorescent, or enzymatic tagthat can be used to detect and/or recover the complex.

Suitable protease inhibitors to use in accordance with the presentinvention include serine protease inhibitors (including immunoglobulinproteinase inhibitors and serpins), metalloprotease inhibitors, asparticacid protease inhibitors, cysteine protease inhibitors andaminopeptidase inhibitors. Preferred protease inhibitors include serineprotease inhibitors, metalloprotease inhibitors, aminopeptidaseinhibitors and cysteine protease inhibitors, particularly those that arebroad spectrum inhibitors. More preferred are broad spectrum serineprotease inhibitors.

There is a wide variety of protease inhibitors, as is known to oneskilled in the art. Examples include, but are not limited to, AEBSF,aprotinin, bestatin, chloromethyl ketones TLCK (Nα-p-tosyl-L-lysinechloromethyl ketone) and TPCK (N-tosyl-L-phenylalanine chloromethylketone), chymostatin, cystatin, 3′4-dichloroisocoumarin, E-64(transepoxysuccinyl-L-leucylamido-(4-guanidino)butane), EDTA(ethylenediaminetetraacetic acid), leupeptin, methyl ketones having avariety of leaving groups, oxidized L-leucinethiol, pepstatin,1,10-orthophenanthroline, phosphoramidon, soybean trypsin/chymotrypsininhibitor and soybean trypsin inhibitor. Preferred protease inhibitorsfor use in the present invention include AEBSF, bestatin, E-64leupeptin, pepstatin, 1,10-orthophenanthroline, phosphoramidon, TLCK andTPCK, with AEBSF (a broad spectrum serine protease inhibitor), bestatin(an inhibitor of leucine aminopeptidase) and 1,10-orthophenanthroline (abroad spectrum metalloprotease inhibitor) being particularly preferred.

Protease inhibitors can be produced using methods known to those skilledin the art. Protein- or peptide-based protease inhibitors, such ascystatin or small peptides comprising a protease substrate, can beproduced recombinantly and modified as necessary.

The present invention also includes the use of proteolytically activeflea protease proteins of the present invention to identify additionalprotease inhibitors, and preferably protease inhibitor compounds thatcan be included in a composition of the present invention to beadministered to animals. A method to identify a flea protease inhibitorincludes the steps of (a) contacting (e.g., combining, mixing) anisolated flea protease protein with a putative (i.e., candidate)inhibitory compound under conditions in which, in the absence of thecompound, the protein has proteolytic activity, and (b) determining ifthe putative inhibitory compound inhibits the proteolytic activity ofthe protein. Putative inhibitory compounds to screen include organicmolecules, antibodies (including functional equivalents thereof) andsubstrate analogs. Methods to determine protease activity are known tothose skilled in the art, as heretofore disclosed. Particularlypreferred for use in identifying inhibitors are flea serine proteaseproteins, flea aminopeptidase proteins and flea cysteine proteaseproteins of the present invention.

The present invention also includes inhibitors isolated by such amethod, and/or test kit, and their use to inhibit any flea protease thatis susceptible to such an inhibitor.

It is to be appreciated that the present invention also includesmimetopes of compounds of the present invention that can be used inaccordance with methods as disclosed for compounds of the presentinvention. As used herein, a mimetope of a proteinaceous compound of thepresent invention (e.g., a flea protease protein, an anti-flea proteaseantibody, a proteinaceous inhibitor of protease activity or synthesis)refers to any compound that is able to mimic the activity of thatproteinaceous compound, often because the mimetope has a structure thatmimics the proteinaceous compound. For example, a mimetope of a fleaprotease protein is a compound that has an activity similar to that ofan isolated flea protease protein of the present invention. Mimetopescan be, but are not limited to: peptides that have been modified todecrease their susceptibility to degradation; anti-idiotypic and/orcatalytic antibodies, or fragments thereof; non-proteinaceousimmunogenic portions of an isolated protein (e.g., carbohydratestructures); and synthetic or natural organic molecules, includingnucleic acids. Such mimetopes can be designed using computer-generatedstructures of proteins of the present invention. Mimetopes can also beobtained by generating random samples of molecules, such asoligonucleotides, peptides or other organic molecules, and screeningsuch samples by affinity chromatography techniques using thecorresponding binding partner.

The present invention includes therapeutic compositions, also referredto herein as compositions, that include a (i.e., at least one) compoundof the present invention. Preferred compounds to include in acomposition of the present invention include flea protease vaccines,anti-flea protease antibodies and/or protease inhibitors as disclosedherein. Such a therapeutic composition can protect an animal from fleainfestation by reducing flea protease activity, thereby reducing fleaburden on the animal and in the environment of the animal.

Particularly preferred therapeutic compositions of the present inventioninclude at least one of the following compounds: an isolated flea serineprotease protein or a mimetope thereof; an isolated flea serine proteasenucleic acid molecule that hybridizes under stringent hybridizationconditions with a flea serine protease gene; an isolated antibody thatselectively binds to a flea serine protease protein; an inhibitor offlea serine protease activity identified by its ability to inhibit fleaserine protease activity; an isolated flea cysteine protease protein ora mimetope thereof; an isolated flea cysteine protease nucleic acidmolecule that hybridizes under stringent hybridization conditions with aflea cysteine protease gene; an isolated antibody that selectively bindsto a flea cysteine protease protein; and an inhibitor of flea cysteineprotease activity identified by its ability to inhibit flea cysteineprotease activity.

Another embodiment of the present invention is a therapeutic compositionthat includes a first compound that reduces flea protease activity and asecond compound that reduces flea burden by a method other than byreducing flea protease activity. The present invention also includes amethod to protect an animal from flea infestation by administering tothe animal such a composition. The first compound of such a compositionby effectively reducing flea protease activity in the midgut, enhancesthe activity of the second compound. While not being bound by theory, itis believed that a number of anti-flea treatments, particularly thosethat are proteinaceous, are not very effective because they are degradedin the flea midgut. The present invention permits the effective use ofsuch anti-flea treatments by reducing proteolytic degradation of suchtreatments by the flea midgut.

Preferred first compounds to include in such a composition include fleaprotease vaccines, anti-flea protease antibodies and/or proteaseinhibitors as disclosed herein, such compounds that target fleaimmunoglobulin proteinase activity.

A preferred therapeutic composition of the present invention comprisesan excipient and a protective compound including: an isolated protein ormimetope thereof encoded by a nucleic acid molecule that hybridizesunder stringent hybridization conditions with a nucleic acid moleculehaving a nucleic acid sequence encoding a protein comprising an aminoacid sequence including SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ IDNO:19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ IDNO:33, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ IDNO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ IDNO:72, SEQ ID NO:73, SEQ ID NO:96, SEQ ID NO:2, SEQ ID NO:5, SEQ IDNO:8, SEQ ID NO:89, SEQ ID NO:92 and/or SEQ ID NO:95; an isolatednucleic acid molecule that hybridizes under stringent hybridizationconditions with a gene comprising a nucleic acid sequence including SEQID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:25. SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ IDNO:32, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ IDNO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:88, SEQ ID NO:90,SEQ ID NO:91, SEQ ID NO:93 and/or SEQ ID NO:94; an isolated antibodythat selectively binds to a protein encoded by a nucleic acid moleculethat hybridizes under stringent hybridization conditions with a nucleicacid molecule having a nucleic acid sequence encoding a proteincomprising an amino acid sequence including SEQ ID NO:10, SEQ ID NO:13,SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27,SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:41,SEQ ID NO:44, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70,SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:96, SEQ ID NO:2, SEQID NO:5, SEQ ID NO:8, SEQ ID NO:89, SEQ ID NO:92 and/or SEQ ID NO:95; aninhibitor of protease activity identified by its ability to inhibit theactivity of a protein encoded by a nucleic acid molecule that hybridizesunder stringent hybridization conditions with a nucleic acid moleculehaving a nucleic acid sequence encoding a protein comprising an aminoacid sequence including SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ IDNO:19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ IDNO:33, SEQ ID NO:36, gs SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ IDNO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ IDNO:72, SEQ ID NO:73, SEQ ID NO:96, SEQ ID NO:2, SEQ ID NO:5, SEQ IDNO:8, SEQ ID NO:89, SEQ ID NO:92 and/or SEQ ID NO:95; and a mixturethereof.

Suitable second compounds include any anti-flea agent(s), including, butnot limited to, proteinaceous compounds, insecticides and flea collars.Preferred second compounds are proteinaceous compounds that effectactive immunization (e.g., antigen vaccines), passive immunization(e.g., antibodies), or that otherwise inhibit a flea activity that wheninhibited can reduce flea burden on and around an animal. Examples ofsecond compounds include a compound that inhibits binding between a fleamembrane protein and its ligand (e.g., a compound that inhibits fleaATPase activity or a compound that inhibits binding of a peptide orsteroid hormone to its receptor), a compound that inhibits hormone(including peptide or steroid hormones) synthesis, a compound thatinhibits vitellogenesis (including production of vitellin and transportand maturation thereof into a major egg yolk protein), a compound thatinhibits fat body function, a compound that inhibits flea muscle action,a compound that inhibits the flea nervous system, a compound thatinhibits the flea immune system and/or a compound that inhibits fleafeeding.

Compositions of the present invention can also include is othercomponents such as a pharmaceutically acceptable excipient, an adjuvant,and/or a carrier. For example, compositions of the present invention canbe formulated in an excipient that the animal to be treated cantolerate.

Examples of such excipients include water, saline, Ringer's solution,dextrose solution, Hank's solution, and other aqueous physiologicallybalanced salt solutions. Nonaqueous vehicles, such as fixed oils, sesameoil, ethyl oleate, or triglycerides may also be used. Other usefulformulations include suspensions containing viscosity enhancing agents,such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipientscan also contain minor amounts of additives, such as substances thatenhance isotonicity and chemical stability. Examples of buffers includephosphate buffer, bicarbonate buffer and Tris buffer, while examples ofpreservatives include thimarosal, m- or o-cresol, formalin and benzylalcohol. Standard formulations can either be liquid injectables orsolids which can be taken up in a suitable liquid as a suspension orsolution for injection. Thus, in a non-liquid formulation, the excipientcan comprise dextrose, human serum albumin, preservatives, etc., towhich sterile water or saline can be added prior to administration.

In one embodiment of the present invention, the composition can alsoinclude an immunopotentiator, such as an adjuvant or a carrier.Adjuvants are typically substances that generally enhance the immuneresponse of an animal to a specific antigen. Suitable adjuvants include,but are not limited to, Freund's adjuvant; other bacterial cell wallcomponents; aluminum-based salts; calcium-based salts; silica;polynucleotides; toxoids; serum proteins; viral coat proteins; otherbacterial-derived preparations; gamma interferon; block copolymeradjuvants, such as Hunter's Titermax adjuvant (Vaxcel™, Inc. Norcross,Ga.); Ribi adjuvants (available from Ribi ImmunoChem Research, Inc.,Hamilton, Mont.); and saponins and their derivatives, such as Quil A(available from Superfos Biosector A/S, Denmark). Carriers are typicallycompounds that increase the half-life of a therapeutic composition inthe treated animal. Suitable carriers include, but are not limited to,polymeric controlled release formulations, biodegradable implants,liposomes, bacteria, viruses, oils, esters, and glycols.

One embodiment of the present invention is a controlled releaseformulation that is capable of slowly releasing a composition of thepresent invention into an animal. As used herein a controlled releaseformulation comprises a composition of the present invention in acontrolled release vehicle. Suitable controlled release vehiclesinclude, but are not limited to, biocompatible polymers, other polymericmatrices, capsules, microcapsules, microparticles, bolus preparations,osmotic pumps, diffusion devices, liposomes, lipospheres, andtransdermal delivery systems. Other controlled release formulations ofthe present invention include liquids that, upon administration to ananimal, form a solid or a gel in situ. Preferred controlled releaseformulations are biodegradable (i.e., bioerodible).

A preferred controlled release formulation of the present invention iscapable of releasing a composition of the present invention into theblood of the treated animal at a constant rate sufficient to attaintherapeutic dose levels of the composition to reduce protease activityin fleas feeding from the animal over a period of time ranging fromabout 1 to about 12 months. A controlled release formulation of thepresent invention is capable of effecting a treatment for preferably atleast about 1 month, more preferably at least about 3 months and evenmore preferably for at least about 6 months, even more preferably for atleast about 9 months, and even more preferably for at least about 12months.

In order to protect an animal from flea infestation, a therapeuticcomposition of the present invention is administered to the animal in aneffective manner such that the protease activity of fleas feeding fromthe blood stream of animals treated with the composition is reduced. Assuch, a treated animal is an animal that is competent to reduce the fleaburden by reducing flea protease activity, or by reducing flea proteaseactivity and at least one other flea activity. Preferably, the proteaseactivity is reduced by at least about 50 percent, more preferably by atleast about 70 percent and even more preferably by at least about 90percent. Methods to administer compositions to the animal in order torender the animal competent depend on the nature of the composition andadministration regime. Animals administered a protease vaccine with atleast one booster shot usually become competent at about the same timeas would be expected for any vaccine treatment. For example, animalsadministered a booster dose about 4 to 6 weeks after a primary doseusually become competent within another about 3 to 4 weeks. Animalsadministered a composition including an anti-flea protease antibody orprotease inhibitor become competent as soon as appropriate serum levelsof the compound are achieved, usually with one to three days.

In a preferred embodiment, a composition of the present invention whenadministered to a host animal is able to reduce flea viability by atleast about 50 percent within at least about 21 days after the fleasbegin feeding from the treated animal. (Note that fleas usually liveabout 40 days to about 50 days on one or more animals.) A more preferredcomposition when administered to a host animal is able to reduce fleaviability by at least about 65 percent within at least about 14 daysafter the fleas begin feeding from the treated animal. An even morepreferred composition when administered to an animal is able to reduceflea viability by at least about 90 percent within at least about 7 daysafter the fleas begin feeding from the treated animal.

In another preferred embodiment, a composition of the present inventionwhen administered to a host animal is able to reduce flea fecundity(i.e., egg laying ability) by at least about 50 percent, more preferablyby at least about 70 percent, and even more preferably by at least about90 percent, within at least about 30 days after the fleas begin feedingfrom the treated animal. (Note that fleas usually do not begin layingeggs until about 7 days after taking a blood meal.)

In accordance with the present invention, compositions are administeredto an animal in a manner such that the animal becomes competent toreduce flea protease activity in a flea that feeds from the competent;i.e., the animal becomes a treated animal. For example, a flea proteasevaccine of the present invention, when administered to an animal in aneffective manner, is able to elicit (i.e., stimulate) an immune responsethat produces an antibody titer in the blood stream of the animalsufficient to reduce flea protease activity. Similarly, an anti-fleaprotease antibody of the present invention, when administered to ananimal in an effective manner, is administered in an amount so as to bepresent in the animal's blood stream at a titer that is sufficient toreduce flea protease activity. A protease inhibitor compound of thepresent invention, when administered to an animal in an effectivemanner, is administered in a manner so as to be present in the animal'sblood stream at a concentration that is sufficient to reduce fleaprotease activity. Oligonucleotide nucleic acid molecules of the presentinvention can also be administered in an effective manner, therebyreducing expression of flea proteases.

Compositions of the present invention can be administered to animalsprior to or during flea infestation. It is to be noted that whenvaccines of the present invention are administered to an animal, a timeperiod is required for the animal to elicit an immune response beforethe animal is competent to inhibit protease activity of fleas feedingfrom that animal. Methods to obtain an immune response in an animal areknown to those skilled in the art.

Acceptable protocols to administer compositions in an effective mannerinclude individual dose size, number of doses, frequency of doseadministration, and mode of administration. Determination of suchprotocols can be accomplished by those skilled in the art. A suitablesingle dose is a dose that is capable of protecting an animal from fleainfestation when administered one or more times over a suitable timeperiod. For example, a preferred single dose of a protease vaccine or amimetope thereof ranges from about 1 tmicrogram (μg, also denoted ug) toabout 10 milligrams (mg) of the composition per kilogram body weight ofthe animal. Booster vaccinations can be administered from about 2 weeksto several years after the original administration. Booster vaccinationspreferably are administered when the immune response of the animalbecomes insufficient to protect the animal from flea infestation. Apreferred administration schedule is one in which from about 10 μg toabout 1 mg of the vaccine per kg body weight of the animal isadministered from about one to about two times over a time period offrom about 2 weeks to about 12 months. In one embodiment, a booster doseof a composition of the present invention is administered about 4 to 6weeks after the primary dose, and additional boosters are administeredabout once or twice a year. Modes of administration can include, but arenot limited to, oral, nasal, topical, transdermal, rectal, andparenteral routes. Parenteral routes can include, but are not limited tosubcutaneous, intradermal, intravenous, and intramuscular routes.

In another embodiment, a preferred single dose of an anti-flea proteaseantibody composition or a mimetope thereof ranges from about 1 μg toabout 10 mg of the composition per kilogram body weight of the animal.Anti-flea antibodies can be re-administered from about 1 hour to aboutbiweekly for several weeks following the original administration.Booster treatments preferably are administered when the titer ofantibodies of the animal becomes insufficient to protect the animal fromflea infestation. A preferred administration schedule is one in whichfrom about 10 μg to about 1 mg of an anti-flea protease antibodycomposition per kg body weight of the animal is administered about every2 to every 4 weeks. Suitable modes of administration are as disclosedherein and are known to those skilled in the art.

According to one embodiment, a nucleic acid molecule of the presentinvention can be administered to an animal in a fashion to enableexpression of that nucleic acid molecule into a protective protein(e.g., flea protease vaccine, anti-flea protease antibody, or.proteinaceous protease inhibitor) or protective RNA (e.g., antisenseRNA, ribozyme or RNA drug) in the animal to be protected from disease.Nucleic acid molecules can be delivered to an animal in a variety ofmethods including, but not limited to, (a) direct injection (e.g., as“naked” DNA or RNA molecules, such as is taught, for example in Wolff etal., 1990, Science 247, 1465-1468) or (b) packaged as a recombinantvirus particle vaccine or as a recombinant cell vaccine (i.e., deliveredto a cell by a vehicle selected from the group consisting of arecombinant virus particle vaccine and a recombinant cell vaccine).

A recombinant virus particle vaccine of the present invention includes arecombinant molecule of the present invention that is packaged in aviral coat and that can be expressed in an animal after administration.Preferably, the recombinant molecule is packaging-deficient. A number ofrecombinant virus particles can be used, including, but not limited to,those based on alphaviruses, poxviruses, adenoviruses, herpesviruses,and retroviruses. When administered to an animal, a recombinant virusparticle vaccine of the present invention infects cells within theimmunized animal and directs the production of a protective protein orRNA nucleic acid molecule that is capable of protecting the animal fromdisease caused by a parasite of the present invention. A preferredsingle dose of a recombinant virus particle vaccine of the presentinvention is from about 1×10⁴ to about 1×10⁷ virus plaque forming units(pfu) per kilogram body weight of the animal. Administration protocolsare similar to those described herein for protein-based vaccines.

A recombinant cell vaccine of the present invention includes recombinantcells of the present invention that express at least one protein of thepresent invention. Preferred recombinant cells include Salmonella, E.coli, Mycobacterium, S. frugiperda, baby hamster kidney, myoblast G8,COS, MDCK and CRFK recombinant cells, with Salmonella recombinant cellsbeing more preferred. Such recombinant cells can be administered in avariety of ways but have the advantage that they can be administeredorally, preferably at doses ranging from about 10⁸ to about 10¹²bacteria per kilogram body weight. Administration protocols are similarto those described herein for protein-based vaccines. Recombinant cellvaccines can comprise whole cells or cell lysates.

Compositions of the present invention can be administered to any animalsusceptible to flea infestation, including warm-blooded animals.Preferred animals to treat include mammals and birds, with cats, dogs,humans, cattle, chinchillas, ferrets, goats, mice, minks, rabbits,raccoons, rats, sheep, squirrels, swine, chickens, ostriches, quail andturkeys as well as other furry animals, pets and/or economic foodanimals, being more preferred. Particularly preferred animals to protectare cats and dogs.

The present invention includes compositions to treat flea infestation byany flea. As such, compositions of the present invention can be derivedfrom any flea species. Preferred fleas to target include fleas of thefollowing genera: Ctenocephalides, Cyopsyllus, Diamanus (Oropsylla),Echidnophaga, Nosopsyllus, Pulex, Tunga, and Xenopsylla, with those ofthe species Ctenocephalides canis, Ctenocephalides felis, Diamanusmontanus, Echidnophaga gallinacea, Nosopsyllus faciatus, Pulex irritans,Pulex simulans, Tunga penetrans and Xenopsylla cheopis being morepreferred. Particularly preferred fleas from which to protect animalsinclude fleas of the species Ctenocephalides felis, Ctenocephalidescanis, and Pulex species (e.g., Pulex irritans and Pulex simulans). Itis also within the scope of the present invention to administercompositions of the present invention directly to fleas.

The present invention also includes the use of compositions of thepresent invention to reduce infestation by other ectoparasites as wellas the use of compositions including protease vaccines, anti-proteaseantibodies and compounds that inhibit protease synthesis and/or activityderived from any actoparasite to reduce actoparasite infestation,particularly controlled release formulations containing suchcompositions. Preferred ectoparasites to target includearachnids,.insects and leeches. More preferred ectoparasites to targetinclude fleas; ticks, including both hard ticks of the family Ixodidae(e.g., Ixodes and Amblyomma) and soft ticks of the family Argasidae(e.g., Ornithodoros, such as O. parkeri and O. turicata); flies, such asmidges (e.g., Culicoides), mosquitos, sand flies, black flies, horseflies, horn flies, deer flies, tsetse flies, stable flies,myiasis-causing flies and biting gnats; ants; spiders, lice; mites; andtrue bugs, such as bed bugs and kissing bugs, including those carryingChagas disease. Even more preferred ectoparasites to target includefleas, mosquitos, midges, sandflies, blackflies, ticks and Rhodnius.

The following examples are provided for the purposes of illustration andare not intended to limit the scope of the present invention.

EXAMPLES

It is to be noted that the Examples include a number of molecularbiology, microbiology, immunology and biochemistry techniques consideredto be known to those skilled in the art. Disclosure of such techniquescan be found, for example, in Sambrook et al., ibid., Borovsky, ArchInsect Biochem. and Phys., 7:187-210, 1988, and related references.Examples 1 through 21, and the the sequence information provided in thesequence listing therein, of related PCT Publication No. WO 96/11706,published Apr. 25, 1996, are incorporated herein by this reference intheir entirety.

Example 1

This example describes the cloning and sequencing of a flea cysteineprotease nucleic acid molecule.

A flea cysteine protease nucleic acid molecule, referred to herein asnfCP1₅₇₃ was produced by PCR amplification using the following method.Primer Cal3F (designed to obtain a calreticulin gene), having nucleicacid sequence 5′ TTG GGA TAC ACT TTG ACT GTT AAC C 3′, representedherein as SEQ ID NO:97 was used in combination with the M13 universalprimer, to PCR amplify, using standard techniques, a DNA fragment from abovine blood-fed whole flea cDNA expression library as described abovein Example 8 of related PCT Publication No. WO 96/11706. Surprisingly,the isolated DNA fragment correlated with a cysteine protease nucleicacid sequence. Sequence from this DNA fragment was used to design primerCys1R, having the nucleic acid sequence 5′ GTG AGC AAC CAT TAT TTC CATATC 3′, represented herein as SEQ ID NO:98, which was used in a secondPCR amplification in combination with the M13 reverse primer. A thirdPCR amplification was performed using primer Cys1F, having the nucleicacid sequence 5′ CTT TCC TCA CAA TAC CAC CAA GGA AGC 3′, representedherein as SEQ ID NO:74, in combination with the M13 universal primer. Afourth PCR amplification was performed using primer Cys2F, having thenucleic acid sequence 5′ CTT GTA CGA TTG TCT CAA CAG GC 3′, representedherein as SEQ ID NO:76, in combination with the M13 universal primer.The resulting PCR products were each gel purified and cloned into the TAVector® System, and subjected to standard DNA sequencing techniques. Acomposite nucleic acid sequence representing a flea cysteine proteasecoding region was deduced, referred to herein as nfCP1₅₇₃, was deducedand is denoted herein as SEQ ID NO:76. Translation of SEQ ID NO:76suggests that nucleic acid molecule nfCP1₅₇₃ encodes a non-full-lengthflea cysteine protease protein of about 191 amino acids, referred toherein as PfCP1₁₉₁, having amino acid sequence SEQ ID NO:77, assumingthe first codon spans from about nucleotide 1 through about nucleotide 3of SEQ ID NO:76.

The nucleic acid and amino acid sequences of the nfCP₅₇₃ nucleic acidmolecule and PfCP1₁₉₁ protein, respectively, were compared to knownnucleic acid and amino acid sequences using a Genbank homology search.SEQ ID NO:77 was found to be similar to the amino acid sequence of P.sativum cysteine protease. The most highly conserved region ofcontinuous similarity between SEQ ID NO:77 and P. sativum cysteineprotease amino acid sequences spans from about amino acid 71 throughabout amino acid 165 of SEQ ID NO:77 and from about amino acid 17through about amino acid 168 of the P. sativum cysteine protease, therebeing about 42% identity between the two regions. Comparison of thenucleic acid sequence encoding amino acids from about 205 through about492 of nfCP1₅₇₃ indicate that those regions are about 54% identical.

Example 2

This example describes the cloning and sequencing of certain flea serineprotease nucleic acid molecules.

Certain serine protease cDNA nucleic acid molecules have been isolatedfrom reverse transcriptase PCR amplification of mRNA isolated from catblood-fed whole fleas. The mRNA was isolated from fleas gathered over 72hours after the initiation of feeding on cat blood. As such, the mRNAcomprised a mixture of mRNA isolated at different time points over 72hours. The mRNA was isolated using ground-up fleas, extracting totalflea RNA using Tri-Reagent (available from Molecular Research Center,Cincinnati, Ohio)and an Invitrogen Fast Track™ RNA isolation kit(available from Invitrogen, Inc. San Diego, Calif.). cDNA wassynthesized using a Stratagene RT-PCR kit (available from Stratagene,Inc, San Diego, Calif.). Primers used for first-strand EDNA synthenisincluded an equal molar mixture of the following: 5′dT-2VT3′ and5′dT-2VC3′ (as provided in a differential display kit, available fromOperon Technologies, Inc. Alameda, Calif.).

The actual primers used in the PCR amplification of the cDNA describedabove included cat-try #2 (SEQ ID NO:86) used in combination with H57primer (SEQ ID NO:99). The resultant PCR products were gel purified andcloned into the TA Vector™. Recombinant TA vector clones were isolatedand the nucleic acid molecules were subjected to nucleic acid sequencingusing analysis as described above.

A. A nucleic acid sequence of a flea serine protease nucleic molecule,namely nfSP24₄₁₀, is represented herein as SEQ ID NO:78. Translation ofSEQ ID NO:78. suggests that nucleic acid molecule nfSP24₄₁₀ encodes anon-full-length flea serine protease protein of about 136 amino acids,referred to herein as PfSP24₁₃₆, having amino acid sequence SEQ IDNO:79, assuming the first codon spans from about nucleotide 1 throughabout nucleotide 3 of SEQ ID NO:78. A Genbank homology search revealedmost homology between SEQ ID NO:79 and an Anopheles gambiae chymotrypsinprotein sequence, there being about 38% identity between correspondingregions of the two amino acid sequences.

B. Another nucleic acid sequence of a flea serine protease nucleicmolecule, namely nfSP33₄₂₆, is represented herein as SEQ ID NO:82.Translation of SEQ ID NO:82 suggests that nucleic acid moleculenfSP33₄₂₆ encodes a non-full-length flea serine protease protein ofabout 142 amino acids, referred to herein as PfSP33₁₄₂, having aminoacid sequence SEQ ID NO:83, assuming the first codon spans from aboutnucleotide 1 through about nucleotide 3 of SEQ ID NO:82. A Cenbankhomology search revealed most homology between SEQ ID NO:83 and aDrosophila serine protease stubble protein sequence, there being about45% identity between corresponding regions of the two amino acidsequences.

Example 3

This example describes the cloning and sequencing of a flea serineprotease nucleic acid molecule.

A serine protease cDNA nucleic acid molecule was isolated in a mannersimilar to that described in Example 8 of related PCT Publication No. WO96/11706. The actual primers used in PCR amplification of the serineprotease nucleic acid molecule from a cat blood-fed whole flea cDNAexpression library (produced as described in Example 2) included cat-try#2 (SEQ ID NO:86) in combination with M13 reverse primer (SEQ ID NO:87).The resulting PCR product was diluted 1:25 and used as a template in asecond PCR reaction using the forward vector primer T3 in combinationwith the reverse primer (derived from the nucleic acid sequence ofnfSP33₇₇₈, described in Example 2) having the nucleic acid sequence 5′ATT CCT CGT GGT TCA GTC GCT C 3′, represented herein as SEQ ID NO:100.The resultant PCR product was gel purified and cloned into the TAVector™. The clones were subjected to nucleic acid sequencing asdescribed above.

A nucleic acid sequence of a flea serine protease nucleic molecule,namely nfSP33₇₇₈ is represented herein as SEQ ID NO:84. As expected, SEQID NO:84 includes a portion of SEQ ID NO:82. Translation of SEQ ID NO:84suggests that nucleic acid molecule nfSP33₇₇₈, encodes a non-full-lengthflea serine protease protein of about 259 amino acids, referred toherein as PfSP33₂₅₉, having amino acid sequence SEQ ID NO:85, assumingthe first codon spans from about nucleotide 2 through about nucleotide 4of SEQ ID NO:84. A Genbank homology search revealed most homologybetween SEQ ID NO:84 and a Drosophila serine protease stubble gene,there being about 54% identity between nucleotides 23-778 of SEQ IDNO:84 and nucleotides 2324-3064 of the Drosophila serine proteasestubble gene.

Example 4

This example describes the cloning and sequencing of certain larval fleaserine protease nucleic acid molecules.

Certain serine protease cDNA nucleic acid molecules have been isolatedfrom a mixed instar larval cDNA library produced using 1st, 2nd and 3rdinstar larvae fed on cat blood, by PCR amplification. The actual primersused in the PCR amplification included either cat-try #2 (SEQ ID NO:86)in combination with either H57 primer (SEQ ID NO:99)or M13 reverseprimer (SEQ ID NO:87). The resultant PCR products were gel purified andcloned into the TA Vector™. Three recombinant TA vector clones wereisolated containing PCR products using cat-try #2 and M13 reverse asprimers and one clone was isolated containing PCR products using cat-try#2 and H57 primers. These newly cloned nucleic acid molecules weresubjected to nucleic acid sequencing as described above. A. A nucleicacid sequence of a larval flea serine protease nucleic molecule isolatedusing cat-try #2 and H57 primers, namely nfSP32₄₃₃, is representedherein as SEQ ID NO:80. Translation of SEQ ID NO:80 suggests thatnucleic acid molecule nfSP32₄₃₃ encodes a non-full-length flea serineprotease protein of about 144 amino acids, referred to herein asPfSP32₁₄₄, having amino acid sequence SEQ ID NO:81, assuming the firstcodon spans from about nucleotide I through about nucleotide 3 of SEQ IDNO:80. A Genbank homology search revealed most homology between SEQ IDNO:80 and an Anopheles gambiae trypsin gene, there being about 52%identity between corresponding regions of the two nucleic acidmolecules.

Example 5

This example describes the isolation and characterization of a 31 kDflea serine protease.

Guts from about 1500 fleas that had been fed on cat blood for about 24hours were dissected in Gut Dissection Buffer (50 mM Tris 8.0, 100 mMCaCl₂). The guts were disrupted by freezing and thawing 4 times,followed by sonication. The resulting extracts were clarified bycentrifugation for 20 minutes at 14,000 rpm in a microfuge at 4° C. Thesupernatant was recovered.

The gut supernatant was loaded onto a 3 ml column comprisingp-aminobenzamidine cross-linked to Sepharose beads (Sigma), previouslyequilibrated in Benzamidine Column Buffer (50 mM Tris 8.0, 100 mM CaCl₂,400 mM NaCl). The supernatant was incubated on the column for about 10min. Unbound protein was slowly washed off the column using BenzamidineColumn Buffer until no protein was detectable by Bradford Assay (BioRad).

Proteases bound to the benzamidine column were then eluted using 10 mlBenzamidine Column Buffer supplemented with 10 mM p-aminobenzamidine(brought to pH 8.0 with NaOH). Proteases in the eluant were concentratedand diafiltered into a volume of about 0.3 ml Gut Dissection Bufferusing a Microcon 3 concentrator (Amicon).

About 120 μl of the concentrated eluant was further concentrated to avolume of about 30 μl. Proteases contained in this concentrate wereresolved by gel electrophoresis on a 14% Tris-Glycine electrophoresisgel (15 μl per lane=approximately 300 gut equivalents per lane). Afterelectrophoresis, the separated proteases were blotted onto a PVDFmembrane using a CAPS buffer (10 mM CAPS pH 11, 0.5 mM DTT) The membranewas stained with Coomassie Brilliant Blue. A dominant protein band ofabout 31 kDa was visualized. The membrane was then used for automatedN-terminal sequencing (described in Example 7 of related PCT Pul-lcationNo. WO 96/11706). A partial N-terminal amino acid sequence of the fleaprotease was determined to be IVGGEDVDISTCGWC (denoted SEQ ID NO:68).

Example 6

This example describes the isolation and characterization of a 31 kDflea serine protease contained in a formulation having IgGase activity(i.e., ability to proteolyze immunoglobulin G proteins).

Cat blood-fed flea gut extracts were prepared and selected on abenzamidine column as described above in Example 5. IgG proteaseactivity was assayed by incubating at 37° C., overnight, the benzamidineeluant with cat immunoglobulin G proteins (IgG) purified on Protein Asepharose. The ability of the flea gut benzamidine eluant to digest catIgG was detected by resolving the samples by gel electrophoresis througha 14% SDS-PAGE gel and silver staining the gel using standard methods.The marked decrease (compared with control samples lacking proteaseactivity) of a 50 kDa band on the silver stained gel, representing catIgG heavy chain, indicated that the benzamidine eluant contains IgGprotease activity.

The benzamidine eluant was then purified on a PolyPropylaspartamidehydrophobic interaction chromatography (HIC) column by applying theeluant to the column in buffer containing 0.1 M KPO₄, pH 6.5 and 2 M(NH₂) SO. Proteases bound to the column were eluted using an ammoniumsulfate gradient of 2 M to 0 M in HIC column buffer. Column fractionswere tested for IgG protease activity using the method described above.Fractions containing IgG protease activity were pooled and applied to aPolyCat cation exchange column in 20 M sodium acetate, pH 6. Theproteins were aluted using a sodium chloride gradient of 0 M to 1 M NaClin 20 M sodium acetate. Fractions eluted from the column were tested forIgG protease activity and then each fraction was resolved byelectrophoresis using SDS-PAGE. Fractions having the highest levels ofIgG protease activity included a protein band that migrated at about 31kDa on the SDS-PAGE gel. Weaker protease activity corresponded to anabout 28 kDa band.

The 31 kDa protein present on the SDS-PAGE gel was used for N-terminalamino acid sequencing using the blotting method described above. Apartial N-terminal amino acid sequence was determined to beIVGGEDVDIST(C)GWQI(S)FQ(S)ENLHF(C)GG(S)IIAPK (denoted herein as SEQ IDNO:69). A comparison of SEQ ID NO:69 and SEQ ID NO:68 (described inExample 5) indicates a single residue difference between the two aminoacid sequences at residue 15 of each sequence (i.e., Q and V,respectively). Since SEQ ID NO:69 correlates with IgGase activity, thedata suggests that the larval protein containing SEQ ID NO:68 has IgGaseactivity.

Example 7

This example describes the cloning and sequencing of a 31 kDa fleaserine protease contained in a formulation having IgGase activity.

A flea protease nucleic acid molecule was isolated from a cat blood-fedwhole flea library (described in Example 2) and a bovine blood-fed wholeflea library (described in Example 8 of related PCT Publication No. WO96/11706) by PCR amplification. The actual primers used in the PCRamplification included FP31A primer designed using the N-terminal aminoacid sequence SEQ ID NO:68, the primer having the nucleic acid sequence5′ GAA GAT GTW GAT ATT TCW ACA TGT GG 3′ (SEQ ID NO:101) used incombination with the M13 universal primer. The resultant PCR productswere gel purified and cloned into the TA Vector™ and subjected tonucleic acid sequencing as described above.

A FP31B primer (5′ GAA AAT GAA ATC CAC TTA AAC ATT ACG 3′), (representedherein as SEQ ID NO:102) was designed using the DNA sequence of a DNAfragment from a bovine blood-fed cDNA library. A flea protease cDNAnucleic acid molecule was isolated by PCR amplification of the catblood-fed whole flea library and the bovine blood-fed whole flea librarydescribed above by PCR amplification. PCR amplification was performedusing the FP31B primer in combination with M13 reverse primer. Theresulting PCR products were then diluted 1:25, and used as a templatefor a second PCR reaction using primer FP31C, having the sequence 5′ CTCTTA TTG TAC GAG GGA TGC 3′ (denoted herein SEQ ID NO:103) in combinationwith T3 primer. The resulting nested PCR product was cloned into TAVector™ and subjected to DNA sequencing.

The nucleic acid sequence of the resulting flea serine protease nucleicmolecule, namely nfSP28₉₂₃ is represented herein as SEQ ID NO:66.Translation of SEQ ID NO:66 yields a protein of about 267 amino acids,denoted PfSP28₂₆₇, having amino acid sequence SEQ ID NO:67, assuming anopen reading frame in which the putative start codon spans from aboutnucleotide 8 through about nucleotide 10 of SEQ ID NO:66 or from aboutnucleotide 11 through about nucleotide 13, and a stop codon spanningfrom about nucleotide 803 through about nucleotide 805 of SEQ ID NO:66.SEQ ID NO:67 contains SEQ ID NO:68 except Q is substituted for C, andSEQ ID NO:69. A Genbank homology search revealed most homology betweenSEQ ID NO:66 and Bombix mori vitellin-degrading protease gene, therebeing about 53% identity between corresponding regions of the twonucleic acid sequences.

Example 8

This example provides additional nucleic acid and deduced amino acidsequences of nucleic acid molecules encoding a flea cysteine proteaseprotein of the present which was described in Example 1. This examplealso provides the production of a cysteine protease protein in E. colicells.

A. Additional Cysteine Protease Nucleic Acid Molecule

The PCR products described in Example 1 were submitted to additionalnucleic acid sequence analysis in order to obtain the nucleic acidsequence of additional portions of the coding region of the cysteineprotease gene. A composite nucleic acid sequence representing a fleacysteine protease coding region, referred to herein as nfCP1₁₁₀₉, wasdeduced and is denoted herein as SEQ ID NO:1. SEQ ID NO:76 is containedwithin the sequence of the nucleic acid molecule nfCP1₁₁₀₉. Translationof SEQ ID NO:1 suggests that nucleic acid molecule nfCP1₁₁₀₉ encodes afull-length flea cysteine protease protein of about 327 amino acids,referred to herein as PfCP1₃₂₇, having amino acid sequence SEQ ID NO:2,assuming an open reading frame in which the initiation codon spans fromabout nucleotide 126 through about nucleotide 128 of SEQ ID NO:1 and thetermination codon spans from about nucleotide 1107 through aboutnucleotide 1109 of SEQ ID NO:1. The complement of SEQ ID NO:1 isrepresented herein by SEQ ID NO:3. The coding region encoding PfCP1₃₂₇,is represented by nucleic acid molecule nfCP1₉₈₄, having a coding strandwith the nucleic acid sequence represented by SEQ ID NO:4 and acomplementary strand with nucleic acid sequence SEQ ID NO:6. Theproposed mature protein, denoted herein as PfCP1₂₂₆, contains about 226amino acids which is represented herein as SEQ ID NO:8. The nucleic acidmolecule encoding PfCP1₂₂₆ is denoted herein as nfCP1₆₈₁, which isrepresented by SEQ ID NO:7. The amino acid sequence of PfCP1₃₂₇ (i.e.,SEQ ID NO:2) predicts that PfCP1₃₂₇ has an estimated molecular weight ofabout 42 kD and an estimated pI of about pI 8.84.

Comparison of nucleic acid sequence SEQ ID NO:1 with nucleic acidsequences reported in GenBank indicates that SEQ ID NO:1 showed the mosthomology, i.e., about 55% identity, with the following three genes: aDrosophila cysteine protease gene, a Bombyx cysteine protease gene and aSarcophaga cysteine protease gene. Comparison of amino acid sequence SEQID NO:2 (i.e., the amino acid sequence of PfCP1₃₂₇) with amino acidsequences reported in GenBank indicates that SEQ ID NO:2 showed the mosthomology, i.e., about 42% identity, with the following three proteins: aDrosophila cysteine protease protein, a Bombyx cysteine protease proteinand a Sarcophaga cysteine protease protein.

B. Production of Cysteine Protease Protein in E. coli cells.

An about 660-bp nucleic acid molecule, referred to herein as nfCP1₆₆₀(designed to encode an apparently mature cysteine protease protein) wasPCR-amplified from a flea mixed instar cDNA library produced using unfed1st instar, bovine blood-fed 1st instar, bovine blood-fed 2^(nd) instarand bovine blood-fed 3^(rd) instar flea larvae (this combination oftissues is referred to herein as mixed instar larval tissues forpurposes of this example). Total RNA was extracted from mixed instartissue using an acid-guanidinium-phenol-chloroform method similar tothat described by Chomczynski et al., 1987, Anal. Biochem. 162, p.156-159. Approximately 5,164 mixed instar larvae were used in each RNApreparation. Poly A+ selected RNA was separated from each total RNApreparation by oligo-dT cellulose chromatography using Poly(A)Quick®mRNA isolation kits (available from Stratagene Cloning Systems, LaJolla, Calif.), according to the method recommended by the manufacturer.A mixed instar cDNA expression library was constructed in lambda (λ)Uni-ZAP™XR vector (available from Stratagene Cloning Systems) usingStratagene's ZAP-cDNA Synthesis Kits protocol. About 6.34 μg of mixedinstar poly A+ RNA were used to produce the mixed instar library. Theresultant mixed instar library was amplified to a titer of about2.17×10¹⁰ pfu/ml with about 97% recombinants. The primers used in thePCR amplification were sense primer CysBS' having the nucleotidesequence 5′ GAT AAG GAT CCG TTA CCA GAT TCT TTC GAC TGG 3′ (containing aBamHI-site; denoted SEQ ID NO:64) and anti-sense primer CysHA having thenucleotide sequence 5′ TTA TCA AGC TTC CAT TTA CAT GCC GTA AAA ATC 3′(containing a HindIII site; denoted SEQ ID NO:65). The resulting PCRproduct nfCP1₆₆₀ was submitted to nucleic acid sequence analysis toobtain a nucleic acid sequence of the coding strand, represented hereinas SEQ ID NO:94. Translation of SEQ ID NO:94 indicated that nfCP1₆₉₆encodes a protein of about 220 amino acids, called PfCP1₂₂₀, having SEQID NO:95. It is to be noted that this sequence analysis indicated thatthe stop codon was actually about 36 base pairs upstream from what hadbeen predicted by SEQ ID NO:1; as such, the protein encoded by nfCP1₆₆₀is about 12 amino acids shorter than would have been predicted by SEQ IDNO:1. The nucleic acid molecule nfCP1₆₆₀ contains the coding region forPfCP1₂₂₀.

Recombinant cell E. coli:pCro-nfCP1₆₆₀ is produced in the followingmanner. Nucleic acid molecule nfCP1₆₆₀ is digested with BamHI andHindIII restriction endonucleases, gel purified, and subcloned intoexpression vector lambdaP_(R)/T²ori/S10HIS-RSET-A9 (the production ofwhich is described in Tripp et al, International PCT Publication No. WO95/24198, published Sep. 14, 1995; see in particular, Example 7), thatis digested with BamHI and HindIII and dephosphorylated. The resultantrecombinant molecule, referred to herein ag pCro-nfCP1₆₆₀, istransformed into E. coli BL-21 competent cells (available from Novagen,Madison, Wis.) to form recombinant cell E. coli:pCro-nfCP1₆₆₀. Therecombinant cell is cultured as described in Example 20 of related PCTPublication No. WO 95/24198. About 1 ml of culture is collected prior toinduction, and about 1 ml of culture is collected about 60 minutesfollowing induction. These samples are then lysed in sodium dodecylsulfate polyacrylamiae gel electrophoresis (SDS-PAGE) loading buffer,resolved on a 14% Tris-glycine acrylamide gel and analyzed by immunoblotusing a T7 (tag) antibody (available from Novagen).

Example 9

This example provides additional nucleic acid and deduced amino acidsequences of nucleic acid molecules encoding serine protease proteins ofthe present invention which are described herein and in the Examplessection of related PCT Publication No. WO 96/11706.

A. A DNA probe labeled with ³²P comprising nucleotides from nfAP2₂₁₀₀(described in Example 23 of related U.S. patent application Ser. No.08/639,075, filed Apr. 24, 1996) was used to screen a bovine blood-fedwhole flea cDNA library (described in Example 8 of related PCTPublication No. WO 96/11706) using standard hybridization techniques. Aclone was isolated having about a 459-nucleotide insert, referred toherein as nfSP18₄₅₉.

A nucleic acid sequence of the composite nucleic acid molecule producedusing nucleic acid sequence from nfSP18₅₃₄ and nfSP18₄₅₉ is referred toherein as nfSP18₇₇₅, having a nucleic acid sequence of the coding strandwhich is denoted herein as SEQ ID NO:9. Translation of SEQ ID NO:9suggests that nucleic acid molecule nfSP18₇₇₅ encodes a non-full-lengthflea serine protease protein of about 228 amino acids, referred toherein as PfSP18₂₂₈, having amino acid sequence SEQ ID NO:10, assumingthe first codon spans from about nucleotide 1 through about nucleotiae 3of SEQ ID NO:9 and the stop coaon spans from about nucleotide 685through about nucleotide 687 of SEQ ID NO:9. The complement of SEQ IDNO:9 is represented herein by SEQ ID NO:11. The coding region encodingPfSP18₂₂₈, is represented by nucleic acid molecule nfSP18₂₂₅, having acoding strand with the nucleic acid sequence represented by SEQ ID NO:12and a complementary strand with nucleic acid sequence SEQ ID NO:14. Theamino acid sequence of PfSP18₂₂₈ (i.e., SEQ ID NO:10) predicts thatPfSP18₂₂₈has an estimated molecular weight of about 25 kD and anestimated pI of about 9.09.

Comparison of nucleic acid sequence SEQ ID NO:9 with nucleic acidsequences reported in GenBank indicates that SEQ ID NO:9 showed the mosthomology, i.e., about 51% identity, between SEQ ID NO:9 and an Anophelesstephensi trypsin 1 gene. Comparison of amino acid sequence SEQ ID NO:10(i.e., the amino acid sequence of PfSP18₂₂₈) with amino acid sequencesreported in GenBank indicates that SEQ ID NO:10 showed the mosthomology, i.e., about 59% identity between SEQ ID NO:10 and Vespa crabroprotein.

B. The remainder of flea serine protease nucleic molecule clone 24(described in Example 2 was determined using primers designed fromnfSP24₄₁₀ to amplify DNA from the bovine blood-fed whole flea cDNAlibrary. Sense primer Flea 24F having the nucleotide sequence 5′ GGA CAAACT GTT CAT TGC AG 3′ (denoted SEQ ID NO:46) was used in combinationwith the M13 universal primer in a first PCR reaction. Anti-sense primerFlea 24R having the nucleotide sequence 5′ CCC TCA TTT GTC GTA ACT CC 3′(denoted SEQ ID NO:47) was used in combination with the M13 reverseprimer in a second PCR reaction. The resulting PCR products were eachgel purified and cloned into the TA Vector® System, and subjected tostandard DNA sequencing techniques.

A composite nucleic acid sequence representing a flea serine proteasecoding region was deduced, referred to herein as nfSP24₁₀₈₉, was deducedand is denoted herein as SEQ ID NO:15. SEQ ID NO:78 is contained withinthe sequence of the nucleic acid molecule nfSP24₁₀₈₉ Translation of SEQID NO:15 suggests that nucleic acid molecule nfSP24₁₀₈₉ encodes afull-length flea serine protease protein of about 258 amino acids,referred to herein as PfSP24₂₅₈, having amino acid sequence SEQ IDNO:16, assuming an open reading frame in which the initiation codonspans from about nucleotide 33 through about nucleotide 35 of SEQ IDNO:15 and the termination codon spans from about nucleotide 807 throughabout nucleotide 809 of SEQ ID NO:15. The complement of SEQ ID NO:15 isrepresented herein by SEQ ID NO:17. The coding region encodingPfSP24₂₅₈, is represented by nucleic acid molecule nfSP24₇₇₄, having acoding strand with the nucleic acid sequence represented by SEQ ID NO:18and a complementary strand with nucleic acid sequence SEQ ID NO:20. Theproposed mature protein, denoted herein as PfSP24₂₃₇, contains about 237amino acids which is represented herein as SEQ ID NO:22. The nucleicacid molecule encoding PfSP24₂₃₇ is denoted herein as nfSP24₇₁₁, whichis represented by SEQ ID NO:21. The amino acid sequence of PfSP24₂₅₈(i.e., SEQ ID NO:16) predicts that PfSP24₂₅₈ has an estimated molecularweight of about 28 kD and an estimated pI of about pI 6.70.

Comparison of nucleic acid sequence SEQ ID NO:15 with nucleic acidsequences reported in GenBank indicates that SEQ ID NO:15 showed themost homology, i.e., about 51% identity between SEQ ID NO:15 and anAnopheles stephensi trypsin 1 gene. Comparison of amino acid sequenceSEQ ID NO:16 (i.e., the amino acid sequence of PfSP24₂₅₈) with aminoacid sequences reported in GenBank indicates that SEQ ID NO:16 showedthe most homology, i.e., about 59% identity between SEQ ID NO:16 and anAnopheles gambiae chymotrypsin II protein.

C. The remainder of flea serine protease nucleic molecule clone 32(described in Example 8 was determined using primers designed fromnfSP32₄₃₃ to amplify DNA from the cat-fed whole flea cDNA library. Senseprimer Flea 32F having the nucleotide sequence 5′ GGC TAG GTT AGT GGATTC TGG 3′ (denoted SEQ ID NO:48) was used in combination with the M13universal primer in a first PCR reaction. Anti-sense primer Flea 32Rhaving the nucleotide sequence 5′ GCA AAT CAG TTC CAG AAT CCA CTA ACC 3′(denoted SEQ ID NO:49) was used in combination with the M13 reverseprimer in a second PCR reaction. The resulting PCR products were eachgel purified and cloned into the TA Vector® System, and subjected tostandard DNA sequencing techniques. .A composite nucleic acid sequencerepresenting a flea serine protease coding region was deduced, referredto herein as nfSP32₉₂₄, was deduced and is denoted herein as SEQ IDNO:23. SEQ ID NO:80 is contained within the sequence of the nucleic acidmolecule nfSP32₉₂₄ Translation of SEQ ID NO:23 suggests that nucleicacid molecule nfSP32₉₂₄ encodes a full-length flea serine proteaseprotein of about 268 amino acids, referred to herein as PfSP32₂₆₈,having amino acid sequence 920 ID NO:24, assuming an open reading framein which the initiation codon spans from about nucleotide 6 throughabout nucleotide 8 of SEQ ID NO:23 and the termination codon spans fromabout nucleotide 810 through about nucleotide 812 of SEQ ID NO:23. Thecomplement of SEQ ID NO:23 is represented herein by SEQ ID NO:25. Thecoding region encoding PfSP32₂₆₉, is represented by nucleic acidmolecule nfSP32₆₉₉, having a coding strand with the nucleic acidsequence represented by SEQ ID NO:26 and a complementary strand withnucleic acid sequence SEQ ID NO:28. The amino acid sequence of PfSP32₂₆₈(i.e., SEQ ID NO:24) predicts that PfSP32₂₆₈ has an estimated molecularweight of about 28.6 kD and an estimated pI of about pI 7.36.

Comparison of nucleic acid sequence SEQ ID NO:23 with nucleic acidsequences reported in GenBank indicates that SEQ ID NO:23 showed themost homology, i.e., about 52% identity between SEQ ID NO:23 and aFusarium oxysporum preprotrypsin gene. Comparison of amino acid sequenceSEQ ID NO:24 (i.e., the amino acid sequence of PfSP32₂₆₈) with aminoacid sequences reported in Gennank indicates that SEQ ID NO:24 showedthe most homology, i.e., about 63% identity between SEQ ID NO:24 and aBombyx mori vitellin -degrading protease precursor protein.

D. The remainder of flea serine protease nucleic molecule clone 33 wasdetermined using primers designed from nfSP33₇₇₈ to amplify DNA from theflea mixed instar larvae cDNA library described above in Example 19.Sense primer Flea 33F having the nucleotide sequence 5′ CAG GGC GCT CTGCAG AAC GCA AC 3′ (denoted SEQ ID NO:50) was used in combination withthe M13 universal primer in a first PCR reaction. Anti-sense primer Flea33R having the nucleotide sequence 5′ ATT CCT CGT GGT TCA GTC GCT C 3′(denoted SEQ ID NO:51) was used in combination with the M13 reverseprimer in a second PCR reaction. The resulting PCR products were eachgel purified and cloned into the TA Vector® System, and subjected tostandard DNA sequencing techniques.

A composite nucleic acid sequence representing a flea serine proteasecoding region was deduced, referred to herein as nfSP33₁₈₉₄, was deducedand is denoted herein as SEQ ID NO:29. SEQ ID NO:84 and SEQ ID NO:82 arecontained within the sequence of the nucleic acid molecule nfSP33₁₈₉₄Translation of SEQ ID NO:29 suggests that nucleic acid moleculenfSP33₁₈₉₄ encodes a full-length flea serine protease protein of about400 amino acids, referred to herein as PfSP33₄₀₀, having amino acidsequence SEQ ID NO:30, assuming an open reading frame in which theinitiation codon spans from about nucleotide 335 through aboutnucleotide 337 of SEQ ID NO:29 and the termination codon spans fromabout nucleotide 1535 through about nucleotide 1537 of SEQ ID NO:29. Thecomplement of SEQ ID NO:29 is represented herein by SEQ ID NO:31. Thecoding region encoding PfSP33₄₀₀, is represented by nucleic acidmolecule nfSP33₁₂₀₀, having a coding strand with the nucleic acidsequence represented by SEQ ID NO:32 and a complementary strand withnucleic acid sequence SEQ ID NO:34. The proposed mature protein, denotedherein as PfSP33₂₄₂, contains about 242 amino acids which is representedherein as SEQ ID NO:36. The nucleic acid molecule encoding PfSP33₂₄₂ isdenoted herein as nfSP33₇₂₆, which is represented by SEQ ID NO:35. Theamino acid sequence of PfSP33₄₀₀ (i.e., SEQ ID NO:30) predicts thatPfSP33₄₀₀has an estimated molecular weight of about 44 kD and anestimated pI of about pI 7.59.

Comparison of nucleic acid sequence SEQ ID NO:29 with nucleic acidsequences reported in GenBank indicates that SEQ ID NO:29 showed themost homology, i.e., about 48% identity between SEQ ID NO:29 and aDrosophila melanogaster serine protease stubble gene. Comparison ofamino acid sequence SEQ ID NO:30 (i.e., the amino acid sequence ofPfSP33₄₀₀) with amino acid sequences reported in GenBank indicates thatSEQ ID NO:30 showed the most homology, i.e., about 63% identity betweenSEQ ID NO:30 and a Drosophila melanogaster serine proteace stubbleprotein.

Example 10

This example provides nucleic acid and deduced amino acid sequence ofanother nucleic acid molecule encoding a serine protease protein of thepresent invention.

A serine protease cDNA nucleic acid molecules has been isolated in amanner similar to that described in Example 8 of related PCT PublicationNo. WO 96/11706. The actual primers used in PCR amplification of theserine protease nucleic acid molecule from a cat blood-fed flea cDNAexpression library (produced as described in Example 8 of related PCTPublication No. WO 96/11706) included cat-try #2 (SEQ ID NO:86) incombination with H57 primer (SEQ ID NO:99). The resultant PCR productwas gel purified and cloned into the TA Vector™. A recombinant TA vectorclone was isolated and subjected to nucleic acid sequencing.

A composite nucleic acid sequence of a flea serine protease nucleicmolecule corresponding to flea clone 40, namely nfSP40₄₂₈ was deducedand is denoted herein as SEQ ID NO:37. Translation of SEQ ID NO:37suggests that nucleic acid molecule nfSP40₄₂₈ encodes a non-full-lengthflea serine proteae protein of about 142 amino acids, referred to hereinas PfSP40₁₄₂, represented herein by SEQ ID NO:38. The complement of SEQID NO:37 is represented herein by SEQ ID NO:39.

The remainder of flea serine protease nucleic molecule clone 40 wasdetermined using primers designed from nfSP40₄₂₈ to amplify DNA from thecat blood-fed whole flea cDNA library. Sense primer Flea 40F having thenucleotide sequence 5′ GGC AAG TTT CGT TTC ACA ATA GG 3′ (denoted SEQ IDNO:52) was used in combination with the M13 universal primer in a firstPCR reaction. Anti-sense primer Flea 40R having the nucleotide sequence5′ TCC AAC CCT AAC TTT TAA ACC TTC 3′ (denoted SEQ ID NO:53) was used incombination with the M13 reverse primer in a second PCR reaction. Theresulting PCR products were each gel purified and cloned into the TAVector® System, and subjected to standard DNA sequencing techniques.

A composite nucleic acid sequence representing a flea serine proteasecoding region was deduced, referred to herein as nfSP40₈₄₁, was deducedand is denoted herein as SEQ ID NO:40. SEQ ID NO:37 is contained withinthe sequence of the nucleic acid molecule nfSP40₈₄₁. Translation of SEQID NO:40 suggests that nucleic acid molecule nfSP40₈₁₄ encodes anon-full-length flea serine protease protein of about 242 amino acids,referred to herein as PfSP40₂₄₂, having amino acid sequence SEQ IDNO:41, assuming an open reading frame in which the first codon spansfrom about nucleotide 2 through about nucleotide 4 of SEQ ID NO:40 andthe termination codon spans from about nucleotide 728 through aboutnucleotide 730 of SEQ ID NO:40. The complement of SEQ ID NO:40 isrepresented herein by SEQ ID NO:42. The coding region encodingPfSP40₂₄₂, is represented by nucleic acid molecule nfSP40₇₁₇, having acoding strand with the nucleic acid sequence represented by SEQ ID NO:43and a complementary strand with nucleic acid sequence SEQ ID NO:45. Theamino acid sequence of PfSP40₂₄₂ (i.e., SEQ ID NO:41) predicts thatPfSP40₂₄₂has an estimated molecular weight of about 26 kD and anestimated pI of about pI 6.5.

Comparison of nucleic acid sequence SEQ ID NO:40 with nucleic acidsequences reported in Genfank indicates that SEQ ID NO:40 showed themost homology, i.e., about 57% identity between SEQ ID NO:40 and aDermatophagoides pteronyssinus Der P3 allergen gene. Comparison of aminoacid sequence SEQ ID NO:41 (i.e., the amino acid sequence of PfSP40₂₄₂)with amino acid sequences reported in Genank indicates that SEQ ID NO:41showed the most homology, i.e., about 40% identity between SEQ ID NO:41and a Bombyx mori vitellin-degrading protease precursor protein.

Example 11

This Example demonstrates the production of serine protease proteins ofthe present invention in E. coli cells.

A. Flea serine protease protein PfSP24₂₅₈ was produced in the followingmanner. An about 714 bp nucleic acid molecule, referred to herein asnfSP24₇₁₄ (designed to encode an apparently mature serine proteaseprotein) was PCR amplified from nfSP24₁₀₈₉ using sense primer Flea 24 EFhaving the nucleotide sequence 5′ CAC AGG ATC CAA TAA TTT GTG GTC AAAATG C 3′ (containing a BamHI-site; denoted SEQ ID NO:54) and anti-senseprimer Flea 24 ER having the nucleotide sequence 5′ AAA AAG AAA GCT TCTTTA ATT TTC TGA CAT TGT CGT G 3′ (containing a HindIII; denoted SEQ IDNO:55). The resulting PCR product nfSP247₁₄ was digested with BamHI andHindIII restriction endonucleases, gel purified, and subcloned intoexpression vector lambdaP_(R)/T²ori/S10HIS-RSET-A9, that had beendigested with BamHI and HindIII and dephosphorylated. The resultantrecombinant molecule, referred to herein as pCro-nfSP24₇₁₄, wastransformed into E. coli BL-21 competent cells (available from Novagen,Madison, Wis.) to form recombinant cell E. coli:pCro-nfSP24₇₁₄. Therecombinant cell was cultured as described in Example 20 of related PCTPublication No. WO 95/24198. About 1 ml of culture was collected priorto induction, and about 1 ml of culture was collected about 60 minutesfollowing induction. These samples were then lysed in sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer andresolved on a 14% Tris-glycine acrylamide gel. Immunoblot analysis ofthe proteins using a T7 (tag) antibody (available from Novagen) showedexpression of an about 36 kD protein in the induced sample but not inthe uninduced sample.

B. Flea serine protease protein PfSP32₂₆₈ was produced in the followingmanner. An about 698 bp nucleic acid molecule, referred to herein asnfSP32₆₉₈ (designed to encode an apparently mature serine proteaseprotein) was PCR amplified from nfSP32₉₃₃ using sense primer Flea 32 EFhaving the nucleotide sequence 5′ GCG GGA TCC TAT TGT GGG TGG TGA AGCAGT G 3′ (containing a BamHI-site; denoted SEQ ID NO:56) and anti-senseprimer Flea 32 ER having the nucleotide sequence 5′ GAC GGT ACC ATG TATAAA ATA ATA TTA AAC TCC GG 3′ (containing a KpnI; denoted SEQ ID NO:57).The resulting PCR product nfSP32₆₉₈ was digested with BamHI and KpnIrestriction endonucleases, gel purified, and subcloned into expressionvector 1 pTrcHisB (available from InVitrogen Corp., San Diego, Calif.),that had been digested with BamHI and KpnI and dephosphorylated. Theresultant recombinant molecule, referred to herein as pTrc-nfSP32₆₉₈,was transformed into E. coli BL-21 competent cells to form recombinantcell E. coli:pTrc-nfSP32₆₉₈. The recombinant cell was cultured andprotein production resolved by SDS-PAGE as described above in Section A.Immunoblot analysis of the proteins using a T7 antibody showedexpression of an about 38 kD protein in the induced sample but not inthe uninduced sample.

C. Flea serine protease protein PfSP33₄₀₀ was produced in the followingmanner. An about 1200 bp nucleic acid molecule, referred to herein asnfSP33₁₂₀₀ (designed to encode an apparently mature serine proteaseprotein) was PCR amplified from nfSP33₁₈₉₄ using sense primer Flea 33 EFhaving the nucleotide sequence 5′ CCG GGA TCC TAT GTT AGC GAT CGT CCCGTC AAA C 3′ (containing a BamHI-site; denoted SEQ ID NO:58) andanti-sense primer Flea 33 ER having the nucleotide sequence 5′ CCG GAATTC TTA TCC CAT TAC TTT GTC GAT CC 3′ (containing a EcoRI; denoted SEQID NO:59). The resulting PCR product nfSP33₁₂₀₀ was digested with BamHIand EcoRI restriction endonucleases, gel purified, and subcloned intoexpression vector lambdaP_(R)/T²ori/S10HIS-RSET-A9, that had beendigested with BamHI and EcoRI and dephosphorylated. The resultantrecombinant molecule, referred to herein as pCro-nfSP33₁₂₀₀, wastransformed into E. coli BL-21 competent cells to form recombinant cellE. coli:pCro-nfSP33₁₂₀₀. The recombinant cell was cultured using themethod described above in Section A.

D. Flea serine protease protein PfSP40₂₄₂ was produced in the followingmanner. An about 716 bp nucleic acid molecule, referred to herein asnfSP40₇₁₆ (designed to encode an apparently mature serine proteaseprotein) was PCR amplified from nfSP40₈₄₁ using sense primer Flea 40 EFhaving the nucleotide sequence 5′ GCG GGA TCC AAT AGT AGG AGG TGA AGATGT AG 3′ (containing a BamHI-site; denoted SEQ ID NO:60) and anti-senseprimer Flea 40 ER having the nucleotide sequence 5′ CCG GAA TTC TTC TAACAA ATT TTA TTT GAT TCC TGC 3′ (containing a EcoRI; denoted SEQ IDNO:61). The resulting PCR product nfSP40₇₁₆ was digested with BamHI andEcoRI restriction endonucleases, gel purified, and subcloned intoexpression vector lambdaP_(R)/T²ori/S10HIS-RSET-A9, that had beendigested with BamHI and EcoRI and dephosphorylated. The resultantrecombinant molecule, referred to herein as pCro-nfSP40₇₁₆₁ wastransformed into E. coli BL-21 competent cells to form recombinant cellE. coli:pCro-nfSP40₇₁₆. The recombinant cell was cultured and proteinproduction resolved using the methods described above in Section A.Immunoblot analysis of the proteins using a T7 antibody showedexpression of an about 38 kD protein in the induced sample but not inthe uninduced sample.

Example 12

This Example demonstrates the production of another serine proteaseprotein of the present invention in E. coli cells.

A. Flea serine protease protein PfSP28₂₃₇ was produced in the followingmanner. An about 711 bp nucleic acid molecule, referred to herein asnfSP28₇₁₁ (designed to encode an apparently mature serine proteaseprotein) was PCR amplified from nfSP28₉₂₃ using sense primer Flea 28 Fhaving the nucleotide sequence 5′ GGA TCC AAT CGT TGG AGG TGA AGA TG 3′(containing a BamHI-site shown in bold; denoted SEQ ID NO:62) andanti-sense primer Flea 28 R having the nucleotide sequence 5′ GAA TTCGAA ATC CAC TTA AAC ATT AGC 3′ (containing a EcoRI shown in bold;denoted SEQ ID NO:63). The resulting PCR product nfSP28₇₁₁ was digestedwith BamHI and EcoRI restriction endonucleases, gel purified, andsubcloned into expression vector lambdaP_(R)/T²ori/S10HIS-RSET-A9, thathad been digested with BamHI and XbaI and dephosphorylated. Theresultant recombinant molecule, referred to herein as pCro-nfSP28₇₁₁,was transformed into E. coli BL-21 competent cells (available fromNovagen, Madison, Wis.) to form recombinant cell E. coli:pCro-nfSP28₇₁₁.The recombinant cell was cultured and protein production resolved usingthe methods described above in Example 21. Immunoblot analysis of theproteins using a T7 antibody showed expression of an about 38 kD proteinin the induced sample but not in the uninduced sample. Immunoblotanalysis using a rabbit anti-flea midgut protease polyclonal antibody(the production of which is described in Example 14 of related PCTPublication NO. WO 95/24198) identified an about 38 kD protein in theinduced sample.

Example 13

This Example demonstrates the production of another serine proteaseprotein of the present invention in eukaryotic cells.

Recombinant molecule pBv-nfSP28₇₉₂, containing a flea serine proteasenucleic acid molecule spanning nucleotides from about 11 through about802 of SEQ ID NO:66, operatively linked to baculovirus polyhedrontranscription control sequences were produced in the following manner.In order to subclone a flea serine protease nucleic acid molecule into abaculovirus expression vector, a flea serine protease nucleic acidmolecule-containing fragment was PCR amplified from nfSP28₉₂₃. A PCRfragment of 792 nucleotides, named nfSP28₇₉₂, was amplified fromnfSP28₉₂₃ using a sense primer Flea 28 F3 having the nucleic acidsequence 5′-GCG GGA TTC TAT AAA TAT GAA ACT TTT GGT AGT TTT TGC -3′ (SEQID NO:62; BamHI site shown in bold) and an antisense primer Flea 28 R3having the nucleic acid sequence 5′-GCT CTA GAC CAC TTA AAC ATT AGC ATATTT TTC-3′ (SEQ ID NO:63; XbaI site shown in bold). The N-terminalprimer was designed from the pol h sequence of baculovirus withmodifications to enhance expression in the baculovirus system.

In order to produce a baculovirus recombinant molecule capable ofdirecting the production of PfSP28₂₆₄, the about 792 base pair PCRproduct (referred to as Bv-nfSP28₇₉₂) was digested with BamHI and XbaIand subcloned into BamHI and XbaI digested to produce the recombinantmolecule referred to herein as pVL-nfSP28₇₉₂.

The resultant recombinant molecule,pVL-nfSP28₇₉₂, was verified forproper insert orientation by restriction mapping. The recombinantmolecule was co-transfected with a linear Baculogold baculovirus DNA(available from Pharmingen) into S. frugiperda Sf9 cells (available fromInVitrogen) to form the recombinant cells denoted S.frugiperda:pVL-nfSP28₇₉₂ . S. frugiperda:pVL-nfSP28₇₉₂ was cultured inorder to produce a flea serine protease protein PfSP28₂₆₄.

Immunoblots of supernatants from cultures of S. frugiperda:pVL-nfSP28₇₉₂cells producing the flea serine protease protein PfSP28₂₆₄ was performedusing a cat anti-fSPFlea 28 polyclonal antibody which was produced asfollows. Recombinant Flea 28 protein (referred to herein as rSPFlea 28protein) produced in E. coli described above in Example 12 was used toimmunize cats. The rSPFlea 28 protein was diluted to a concentration ofabout 1 mg/ml in PBS and emulsified in an equal volume of TiterMaxresearch adjuvant (available from CytRx Corp., Norcross, Ga.). A seriesof cats were immunized each with about 50 μg of rSPFlea 28 protein inadjuvant by subcutaneous injection. A second injection of the same doseof rSPFlea 28 protein in adjuvant was administered 32 days later. Bloodsamples were obtained prior to immunization (pre-bleed), 32 days and 47days after the initial immunization. Sera samples from thepre-immunization and Day 47 bleeds were used for subsequent immunoblotexperiments. The latter is referred to as anti-fSPFlea 28 polyclonalantibody.

Analysis of the immunoblots identified an about 33 kD protein and anabout 36 kD protein.

Example 14

This example describes the production of peptides from the 31 kD fleamidgut serine protease and the generation of internal sequence data.

Midguts from about 30,000 cat blood-fed fleas were dissected asdescribed in U.S. Pat. No. 5,356,622, ibid. in gut dissection buffer (50mM Tris 8.0, 100 mM CaCl₂). The guts (in three batches of about 10,000each) were disrupted by a freeze-thaw cycle, followed by sonication. Theresulting extracts were clarified by centrifugation for 20 minutes at3050 rpm in a swinging bucket centrifuge at 4° C. The supernatants wererecovered, and adjusted to 400 mM NaCl in preparation for benzamidinecolumn chromatography.

For each batch, gut supernatants were loaded into a 5 ml disposablecolumn containing p-aminobenzamidine cross-linked to Sepharose beads(available from Sigma, St. Louis, Mo.), previously equilibrated inbenzamidine column buffer (50 mM Tris, pH 8.0, 100 mM CaCl₂, 400 mMNaCl) and incubated with rocking overnight at 4° C. Unbound protein wasslowly washed off the column using benzamidine column buffer until noprotein was detectable by Bradford Assay (available from Bio-RadLaboratories, Hercules, Calif.).

Proteases bound to the benzamidine column were eluted using 4 mlbenzamidine column buffer supplemented with 100 mM p-aminobenzamidine(brought to pH 8.0 with NaOH). Residual bound proteases were washed offwith about 21 ml of additional benzamidine column buffer. The recoveredproteases were then concentrated to a volume of about 2 ml using aUltrafree 20 10-kD centrifugal concentrator (available from Millipore,Bedford, Mass.). After concentration, the protease pools from the 3preparations were combined for a total of about 30,000 gut equivalentsin about 6 ml. Protein concentration was measured by Bradford assay andfound to be about 0.5 mg/ml.

About 150 μg of the isolated protease pool was resolved bypolyacrylamide gel electrophoresis (PAGE) on a preparative-well 14%Tris-glycine gel (available from Novex, San Diego, Calif.). Afterelectrophoresis, the proteins in the gel were visualized by staining forabout 30 minutes in Coomassie brilliant blue stain (0.1% (w/v) Coomassieblue R, 40% (v/v) methanol, 10% (v/v) acetic acid) and destaining forabout 2.5 hours in 50% (v/v) methanol. The band corresponding to the31-kD protease was excised with a razor blade. The protein waselectroeluted, concentrated, and partially digested for 24 hours withcyanogen bromide (CNBr) (Silver, et al., 1995, J. Biol. Chem., 270,13010-13016), except that a small amount of acetic acid was added to thesample after electroelution and concentration to lower the sample pH andtherefore reduce autodigestion by the 31-kD protease. CNBr is known tocleave after methionine (M) residues under the conditions used for thisdigestion. After CNBr digestion, the peptides in the sample wereresolved by PAGE on an 18% Tris-glycine gel. After electrophoresis, theseparated protease peptides were electroblotted onto a PVDF membraneusing a CAPS buffer (10 mM CAPS pH 11, 0.5 mM DTT, 10% (v/v) methanol).The membrane was stained with Coomassie Brilliant Blue and destainedwith 50% (v/v) methanol. Three stained peptide bands were identifiedhaving apparent molecular weights of about 14 kD, 21 kD, and 22 kD,respectively. The portions of the membrane containing the 21 kD and 22kD bands were excised separately. Peptides contained in each membranesegment were subjected to N-terminal amino acid sequencing using a 473AProtein Sequencer (available from Applied Biosystems, Foster City,Calif.) using standard techniques.

Although the results from the automated sequencing were difficult tointerpret due to overlapping sequences, analysis of the chromatogramsindicated the N-terminal amino acid sequence of the 21-kD peptide to beH/R-V/P-G/A/S-Y/G-E/N-D/K-V/R-D/A-D-Y-D-F-D/P-V-A, denoted herein as SEQID NO:70 and the N-terminal amino acid sequence of the 22-kD peptide tobe I/Q-V-G-Y/G-E/N/T-D/M/P-V-K/D-I-N/S-M/T/N-F/C herein denoted as SEQID NO:71. The N-terminal amino acid sequence of the intact 31-kDprotease is either I-V-G-G-E-D-V-D-I-S-T-C-G-W-C (SEQ ID NO:59, asdisclosed in Example 34 in co-pending U.S. patent application Ser. No.08/639,075), or IVGGEDVDIST(C)GWQI(S)FQ(S)ENLHF(C)GG(S)IIAPK (SEQ IDNO:69, as disclosed in Example 35 in co-pending U.S. patent applicationSer. No. 08/639,075). These sequences vary at residue 15 in that SEQ IDNO:68 contains a cysteine and SEQ ID NO:69 contains a glutamine. Thesesequences can be identified in the sequences of both the 21-kD (SEQ IDNO:70) and 22-kD (SEQ ID NO:71) peptides, though it is much stronger inthe 22-kD peptide, leading to the conclusion that the SEQ ID NO:71represents the N-terminus of the 31-kD protease. If this sequence issubtracted from the sequence for the 21-kD (SEQ ID NO:70) peptide, thenthe resulting sequence for the 21-kD peptide isH/R-P-A/S-Y-N-K-R-A-D-Y-D-F-D-V-A, denoted herein as SEQ ID NO:72. Thissequence of amino acids aligns with a stretch of deduced amino acidsfrom about residue 107 to residue 121 immediately following a methionineresidue in SEQ ID NO:67. These data confirm that the clone representedby nfSP28₉₂₃ (SEQ ID NO:66, as disclosed in Example 36 in co-pendingU.S. patent application Ser. No. 08/639,075) indeed encodes the 31-kDprotease.

Example 15

This example demonstrates that a 31-kD flea midgut serine proteasecontained in a formulation is able to proteolyze cat immunoglobulin G,A, and M proteins as well as bovine, dog, human, and rabbitimmunoglobulin G proteins.

The 31-kD flea midgut serine protease was purified from cat blood-fedfleas as follows. Cat blood-fed flea midgut extracts were prepared andselected on a benzamidine column as described above in Example 25. Thebenzamidine eluate was then further purified as described in Example 35of co-pending U.S. patent application Ser. No. 08/639,075 by PolyCAT Acation exchange chromatography (available from PolyLC, Inc., Columbia,Md.) to isolate a protein band which migrated at about 31 kD on a silverstained SDS-PAGE gel.

A. The ability of the cat blood-fed 31-kD flea midgut serine protease todegrade immunoglobulin was demonstrated by measuring digestion ofimmunoglobulin heavy chain using a method similar to that described inExample 35 of co-pending U.S. patent application Ser. No. 08/639,075.Specifically, 1 μg samples of cat IgG, cat IgA, and cat IgM substrates(available from Bethyl Laboratories, Inc., Montgomery, Tex.) wereincubated with 500 cat blood-fed flea midgut equivalents of purified31-kD flea midgut serine protease in a total volume of 27 μl 0.1MTris-HCl pH 8.0 at 37° C. for 18 hours. The reaction mixtures wereresolved by 14% Tris-glycine SDS-PAGE and the gel was silver stainedusing standard methods. The total disappearance (compared with controlsamples lacking addition of the purified 31-kD protein) of bandsmigrating at about 50, 60, and 80 kD on the silver stained gel in thelanes containing 31-kD protease-treated cat IgG, IgA, and IgM,respectively, indicated that the 31-kD flea midgut serine proteasedegraded the heavy chains of the various cat immunoglobulin isotypes.

B. The ability of the cat blood-fed 31-kD flea midgut serine protease todegrade IgG from several species was demonstrated by incubating 1 μgsamples of purified cat or bovine IgG (purified from cat and bovineblood on Protein A Sepharose), or of purified dog, rabbit, or human IgG(each available from Sigma Chemical Co.) with 500 cat blood-fed fleamidgut equivalents of the purified 31-kD flea midgut serine protease ina total volume of 27 μl 0.1M Tris-HCl pH 8.0 at 37° C. for 18 hours. Thereaction mixtures were resolved by 14% Tris-glycine SDS-PAGE and the gelwas silver stained using standard methods. The total disappearance(compared with control samples lacking addition of the purified 31-kDprotein) of bands migrating at about 50-55-kD on the silver stained gelin the lanes containing the 31-kD protease treated cat, bovine, dog,rabbit, and human IgG heavy chains, indicated that the 31-kD flea midgutserine protease can degrade IgG from various mammalian species.

Example 16

This example describes the ability of a 31-kD flea midgut serineprotease contained in a formulation to proteolyze cat immunoglobulin Gat a specific site.

The 31-kD flea midgut serine protease was purified from cat blood-fedflea midgut extracts as described above in Examples 14 and 15

To investigate cleavage site specificity of the purified 31-kD fleamidgut serine protease, 10 μg of cat immunoglobulin G purified from catblood on Protein A sepharose was incubated with 200 cat blood-fed fleamidgut equivalents of purified 31-kD flea midgut serine protease in atotal volume of 100 μl 0.2 M Tris-HCl pH 8.0 at 17° C. for 18 hours. Thereaction mixture was resolved by 14% Tris-glycine SDS-PAGE, blotted ontoa PVDF membrane, stained with Coomassie R-250 and destained according tostandard procedures. A band of about 33 kD was excised and subjected toN-terminal amino acid sequencing using techniques known to those skilledin the art. A partial N-terminal amino acid sequence of about 28 aminoacids was determined and is represented herein as SEQ ID NO: 73:X-P-P-P-E-M-L-G-G-F-S-I-F-I-F-P-P-K-F-K-D-D-L-L-I-K-R-K. A Genankhomology search using SEQ ID NO:73 revealed most homology to Oxyctolaguscaniculus gamma H-chain constant region 2, there being about 71%identity over the 28 amino acids. Further alignments of SEQ ID NO:73with sheep, rat, rabbit, monkey, bovine, and human IgG amino acidsequences indicated that the purified cat blood-fed 31-kD flea midgutserine protease cleaved the cat IgG heavy chain just before thepredicted C-terminal end of the IgG hinge region. The predicted firstamino acid cysteine and the second amino acid proline occur within thepredicted hinge region while the remaining 26 amino acids of SEQ IDNO:73, starting with the third amino acid proline, occur within thepredicted constant heavy chain-2 region.

The further investigate the cleavage site specificity of the purified31-kD flea midgut serine protease for cat IgG, the cleavage site wascompared to that of a known protease, papain, as follows. Catimmunoglobulin G (100 mg), purified from cat blood on Protein Asepharose, was incubated with 1 mg papain in 100 mM sodium acetate pH5.5, 50 mM cysteine, 1 mM EDTA in a final volume of 150 μl at roomtemperature for 4.5 hours. The reaction mixture was resolved on a 14%Tris-glycine SDS-PAGE gel, blotted onto PVDF membrane, stained withCoomassie R-250 and destained according to standard procedures. A bandof about 33 kD was excised and subjected to N-terminal amino acidsequencing using techniques known to those skilled in the art. A partialN-terminal amino acid sequence of about 25 amino acids was deduced andis represented herein as SEQ ID NO: 96:X-P-P-P-E-M-L-G-G-P-S-I-F-I-F-P-P-K-K-K-D-D-L-L-I. This sequence wasnearly identical to the one obtained from a 33-kD cat IgG cleavageproduct generated by the purified 31-kD flea midgut serine protease, theonly difference being the substitution of a lysine (SEQ ID NO:73) for aproline (SEQ ID NO:96) at amino acid 19.

Example 17

This Example demonstrates the kinetics of cat IgG degrading activity inthe midguts of fleas fed on live cats.

A. To determine the kinetics of cat IgG degradation in the guts ofcontinuously feeding fleas, female fleas contained in chambers were fedon seven separate cats (i.e. one chamber per cat) as described inExample 21 of co-pending U.S. patent application Ser. No. 08/639,075.Flea chambers were removed for dissections at timepoints of 15 min., 30min., 1 hr., 2 hr., 4 hr., 6 hr., 8 hr., and 17 hr. After feeding on thecats, the fleas′ midguts were removed as described in U.S. Pat. No.5,356,622, ibid., homogenized by freeze-fracture and sonicated in a Trisbuffer comprising 50 mM Tris, pH 8.0 and 100 mM CaCl₂. The extracts werecentrifuged at about 14,000×g for 20 min. and the soluble materialrecovered. The soluble material was then diluted to a finalconcentration of about 1.2 midgut equivalents per microliter (μl) ofTris buffer. The proteins contained in 1 midgut equivalent of eachtimepoint were then resolved by SDS-PAGE under reducing conditions, andthe proteins visualized by silver staining. The results indicated thatIgG heavy chain levels were significantly lower at the 17 hour timepoint than in the 8 hour and earlier time points, and that light chainlevels were reduced but not to the same extent as the heavy chain. Theproteins contained in 5 gut equivalents of each timepoint were thenresolved by SDS-PAGE gel under reducing conditions and were subjected towestern blot analysis using alkaline phosphatase labeled goat anti-catIgG (heavy plus light chain) antibody (available from Kirkegaard andPerry Laboratories, Gaithersburg, Md.). The results indicated that catIgG heavy chain was present in the midguts of continuously feeding fleasfor at least 8 hours, but was not detected in the midguts of fleasallowed to feed continuously on a cat for 17 hours. Light chain wasvisible in all samples, though the amount visible in the 17 hour samplewas significantly less than that visible in the 8 hour sample. Theseresults suggest that even when fleas are continuously feeding on a cat,the levels of IgG-degrading proteases induced in the flea midguts at atime point of 17 hours is sufficient to degrade all detectable cat IgGingested. These results suggest that when fleas are continuously feedingon a cat, the levels of IgG-degrading proteases induced in the fleamidguts are not sufficient to degrade all detectable cat IgG ingestedfor at least 8 hours.

B. To determine the kinetics of cat IgG degradation in the guts of fleasfed for a specified time then removed from the cat, fleas (in chambers)were fed on cats as in Section A for periods of either 1 hour or 24hours. Following the 1 or 24-hour feeding periods, the flea chamberswere removed and placed in a 28° C., 75% relative humidity growthincubator. Fleas were subjected to dissection at time points of 0, 1, 2,4, and 8 hr. following removal from the cats. Midguts were homogenized,and the midgut contents were examined by silver stained SDS-PAGE andimmunoblot analysis, as described in Section A. The fleas fed for 1 hourhad high molecular weight proteins, including the heavy chain and lightchain of cat IgG detectable in their midguts at the 0 and 1 hourdissection timepoints, while the flea midgutg evaluated at time pointsof 2 hours or greater had no detectable IgG heavy chain bands. Theresults showed that when fleas were fed on a cat and then removed, theydegraded the ingested cat IgG heavy chain nearly completely within 2hours. The fleas fed on cats for 24 hours had no detectable IgG heavy orlight chain proteins in midgut extracts at any of the timepoints. Theseresults suggest that when no new cat IgG is ingested, as is the casewhen the fleas are removed from feeding, that the IgG-degradingproteases in the flea midgut fully degraded all cat IgG heavy chain inless than two hours.

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims.

What is claimed is:
 1. An isolated nucleic acid molecule comprising aconsecutive 18 nucleotide portion identical in sequence to a consecutive18 nucleotide portion of a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93 and SEQ IDNO:94, wherein said nucleic acid molecule is not mammalian.
 2. Thenucleic acid molecule of claim 1, wherein said nucleic acid sequenceencodes an amino acid sequence selected from the group consisting of SEQID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:89, SEQ ID NO:92 and SEQ IDNO:95.
 3. The nucleic acid molecule of claim 1, wherein said nucleicacid molecule comprises a nucleic acid sequence that encodes a fleaprotease protein selected from the group consisting of a larval cysteineprotease protein and an adult cysteine protease protein.
 4. The nucleicacid molecule of claim 1, wherein said nucleic acid molecule is is aflea nucleic acid molecule.
 5. The nucleic acid molecule of claim 1,wherein said nucleic acid molecule is selected from the group consistingof Ctenocephalides, Ceratophyllus, Diamanus, Echidnophaga, Nosopsyllus,Pulex, Tunga, Oropsylla, Orchopeus and Xenopsylla nucleic acidmolecules.
 6. The nucleic acid molecule of claim 1, wherein said nucleicacid molecule is selected from the group consisting of Ctenocephalidesfelis, Ctenocephalides canis, Ceratophyllus pulicidae, Pulex irritans,Oropsylla (Thrassis) bacchi, Oropsylla (Diamanus) montana, Orchopeushowardi, Xenopsylla cheopis and Pulex simulans nucleic acid molecules.7. The nucleic acid molecule of claim 1, wherein said nucleic acidmolecule comprises a Ctenocephalides felis nucleic acid molecule.
 8. Thenucleic acid molecule of claim 1, wherein said nucleic acid moleculecomprises a nucleic acid molecule selected from the group consisting ofnfCP1₅₇₃ and nfCP1₁₁₀₉.
 9. The nucleic acid molecule of claim 1, whereinsaid nucleic acid molecule is selected from the group consisting of: anucleic acid molecule comprising a nucleic acid sequence that encodes aprotein having an amino acid sequence selected from the group consistingof SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:89, SEQ ID NO:92 andSEQ ID NO:95; and a nucleic acid molecule comprising an allelic variantof a nucleic acid molecule encoding any of said amino acid sequences.10. The nucleic acid molecule of claim 1, wherein said nucleic acidmolecule is selected from the group consisting of a nucleic acidmolecule comprising a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93 and SEQ IDNO:94; and a nucleic acid molecule comprising an allelic variant of anucleic acid molecule having any of said nucleic acid sequences.
 11. Thenucleic acid molecule of claim 1, wherein said nucleic acid moleculecomprises an oligonucleotide.
 12. The nucleic acid molecule of claim 3,wherein said protein, when administered to an animal elicits an immuneresponse against a flea cysteine protease.
 13. A recombinant moleculecomprising a nucleic acid molecule as set forth in claim 1 operativelylinked to a transcription control sequence.
 14. A recombinant viruscomprising a nucleic acid molecule as set forth in claim
 1. 15. Arecombinant cell comprising a nucleic acid molecule as set forth inclaim 1, said cell being capable of expressing said nucleic acidmolecule.
 16. An isolated nucleic acid molecule having a consecutive 18nucleotide portion identical in sequence to a consecutive 18 nucleotideportion of a nucleic acid sequence encoding a protein comprising anamino acid sequence selected from the group consisting of SEQ ID NO:2,SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:89, SEQ ID NO:92 and SEQ ID NO:95,wherein said nucleic acid molecule is not mammalian.
 17. The nucleicacid molecule of claim 16, wherein said nucleic acid molecule comprisesa nucleic acid sequence that encodes a flea protease protein selectedfrom the group consisting of a larval cysteine protease protein and anadult cysteine protease protein.
 18. The nucleic acid molecule of claim16, wherein said nucleic acid molecule is a flea nucleic acid molecule.19. The nucleic acid molecule of claim 16, wherein said nucleic acidmolecule is selected from the group consisting of Ctenocephalides,Ceratophyllus, Diamanus, Echidnophaga, Nosopsyllus, Pulex, Tunga,Oropsylla, Orchopeus and Xenopsylia nucleic acid molecules.
 20. Thenucleic acid molecule of claim 16, wherein said nucleic acid molecule isselected from the group consisting of Ctenocephalides felis,Ctenocephalides canis, Ceratophyllus pulicidae, Pulex irritans,Oropsylla (Thrassis) bacchi, Oropsylla (Diamanus) montana, Orchopeushowardi, Xenopsylla cheopis and Pulex simulans nucleic acid molecules.21. The nucleic acid molecule of claim 16, wherein said nucleic acidmolecule comprises a Ctenocephalides felis nucleic acid molecule. 22.The nucleic acid molecule of claim 16, wherein said nucleic acidmolecule is selected from the group consisting of: a nucleic acidmolecule comprising a nucleic acid sequence encoding a proteincomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:89, SEQ ID NO:92 andSEQ ID NO:95; and a nucleic acid molecule comprising an allelic variantof a nucleic acid sequence encoding a protein having any of said aminoacid sequences.
 23. A recombinant molecule comprising a nucleic acidmolecule as set forth in claim 16 operatively linked to a transcriptioncontrol sequence.
 24. A recombinant virus comprising a nucleic acidmolecule as set forth in claim
 16. 25. A recombinant cell comprising anucleic acid molecule as set forth in claim
 16. 26. A therapeuticcomposition that, when administered to an animal, reduces fleainfestation, said therapeutic composition comprising an isolated nucleicacid molecule having a consecutive 18 nucleotide portion identical insequence to a consecutive 18 nucleotide portion of a nucleic acidsequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:88, SEQ ID NO:90, SEQID NO:91, SEQ ID NO:93 and SEQ ID NO:94, wherein said nucleic acidmolecule is riot mammalian.
 27. The composition of claim 26, whereinsaid composition further comprises a component selected from the groupconsisting of an excipient, an adjuvant, a carrier, and a mixturethereof.
 28. The composition of claim 26, wherein said compositioncomprises a controlled release formulation.
 29. The composition of claim26, wherein said composition further comprises a compound that reducesflea burden by a method other than by reducing flea protease activity.30. A method to produce a flea protease protein, said method comprisingculturing a cell capable of expressing said protein, said protein beingencoded by a nucleic acid molecule having a consecutive 18 nucleotideportion identical in sequence to a consecutive 18 nucleotide portion ofa nucleic acid sequence selected from the group consisting of SEQ IDNO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:88, SEQ ID NO:91 and SEQ IDNO:94, wherein said nucleic acid molecule is not mammalian.